Single domain VHH antibodies against von Willebrand factor

ABSTRACT

The present invention relates to improved Nanobodies™ against von Willebrand Factor (vWF), as well as to polypeptides comprising or essentially consisting of one or more of such Nanobodies. The invention also relates to nucleic acids encoding such Nanobodies and polypeptides; to methods for preparing such Nanobodies and polypeptides; to host cells expressing or capable of expressing such Nanobodies or polypeptides; to compositions comprising such Nanobodies, polypeptides, nucleic acids or host cells; and to uses of such Nanobodies, such polypeptides, such nucleic acids, such host cells or such compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/EP2006/004773, filed May 19, 2006, andclaims the benefit under 35 U.S.C. §119(e) of U.S. provisionalapplication Ser. No. 60/683,474, filed May 20, 2005.

The present invention relates to improved Nanobodies™ against vonWillebrand Factor (vWF), as well as to polypeptides comprising oressentially consisting of one or more of such Nanobodies. [Note:Nanobody™, Nanobodies™ and Nanoclone™ are trademarks of Ablynx N.V.]

The invention also relates to nucleic acids encoding such Nanobodies andpolypeptides; to methods for preparing such Nanobodies and polypeptides;to host cells expressing or capable of expressing such Nanobodies orpolypeptides; to compositions comprising such Nanobodies, polypeptides,nucleic acids or host cells; and to uses of such Nanobodies, suchpolypeptides, such nucleic acids, such host cells or such compositions,in particular for prophylactic, therapeutic or diagnostic purposes, suchas the prophylactic, therapeutic or diagnostic purposes mentioned below.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description hereinbelow.

WO 04/062551 of Applicant relates to Nanobodies against Von WillebrandFactor (vWF) and to the preparation and use thereof, in particular forthe prevention and/or treatment of diseases and disorders relating toplatelet-mediated aggregation.

The anti-vWF Nanobodies according to WO 04/062551 may be humanized andmay be monovalent or multivalent, the latter of which leads to increasedaffinity for vWF. The anti-vWF Nanobodies according to WO 04/062551 mayalso be multispecific, and may in particular be in the form of amultispecific construct comprising two or more Nanobodies against vWFand a further Nanobody directed against a serum protein such as humanserum albumin, which leads to an increased half-life in vivo.

The anti-vWF Nanobodies described in WO 04/062551 may be directedagainst any epitope or conformation of vWF (such as the A1 domain or A3domain), but are preferably directed against the A1 domain, and inparticular against the activated conformation of the A1 domain.

WO 04/062551 also describes the preparation of the anti-vWF Nanobodies,nucleotide sequences encoding the anti-vWF Nanobodies, as well aspharmaceutical compositions comprising the anti-vWF Nanobodies.

The anti-vWF Nanobodies and compositions described in WO 04/062551 maybe used for the prevention and treatment of diseases and disordersrelated to platelet-mediated aggregation, such as the formation of anon-occlusive thrombus, the formation of an occlusive thrombus, arterialthrombus formation, acute coronary occlusion, peripheral arterialocclusive disease, restenosis and disorders arising from coronaryby-pass graft, coronary artery valve replacement and coronaryinterventions such angioplasty, stenting or atherectomy, hyperplasiaafter angioplasty, atherectomy or arterial stenting, occlusive syndromein a vascular system or lack of patency of diseased arteries, thromboticthrombocytopenic purpura (TTP), transient cerebral ischemic attack,unstable or stable angina pectoris, cerebral infarction, HELLP syndrome,carotid endarterectomy, carotid artery stenosis, critical limbischaemia, cardioembolism, peripheral vascular disease, restenosis andmyocardial infarction.

The pharmaceutical compositions described in WO 04/062551 may besuitable for intravenous, subcutaneous, oral, sublingual, topical,nasal, vaginal or rectal administration, or for administration byinhalation; and may also comprise a trombolytic agent, such asstaphylokinase, tissue plasminogen activator, streptokinase, singlechain streptokinase, urokinase and acyl plasminogen streptokinasecomplex. The anti-vWF Nanobodies described in WO 04/062551 may also beused for diagnostic purposes (optionally in the form of a kit-of-parts)or in coatings for medical devices such as stents

It is a general object of the present invention to provide Nanobodiesagainst vWF, in particular against human vWF.

In particular, it is an object of the present invention to provideNanobodies against vWF, in particular against human vWF, and to provideproteins or polypeptides comprising the same, that are suitable fortherapeutic and/or diagnostic use, and in particular for the prevention,treatment and/or diagnosis of one or more diseases and disordersassociated with and/or mediated by vWF such as those mentioned above,and/or that can be used in the preparation of a pharmaceuticalcomposition for the prevention and/or treatment of one or more diseasesassociated with and/or mediated by vWF, such as those mentioned above.

More in particular, it is an object of the invention to provideNanobodies against vWF, and to provide proteins and polypeptidescomprising the same, that are either an alternative to the Nanobodiesand polypeptides against vWF described in WO 04/062551 and/or that haveone or more improved properties or characteristics, compared to theNanobodies and polypeptides against vWF described in WO 04/062551.

More in particular, it is an object of the invention to provideNanobodies against vWF, and to provide proteins or polypeptidescomprising the same, that are improved compared to the Nanobodies andpolypeptides against vWF described in WO 04/062551 with respect to oneor more of the following properties or characteristics:

-   -   increased affinity for vWF, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described in WO 04/062551 or hereinbelow);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described in WO        04/062551 or hereinbelow);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein); and/or    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described in WO 04/062551 or hereinbelow) in a        monovalent format;    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described in WO 04/062551 or hereinbelow) in a        monovalent format;    -   increased specificity towards vWF, either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described in WO 04/062551 or hereinbelow)        in a monovalent format;    -   decreased or where desired increased cross-reactivity with vWF        from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described in WO 04/062551 or hereinbelow).

These objects are achieved by the Nanobodies against vWF and by thepolypeptides described herein. The Nanobodies against vWF andpolypeptides described herein are in particular directed against humanvWF, but it is included within the scope of the invention that some ofthe anti-vWF Nanobodies and polypeptides of the invention may showcross-reactivity with vWF from other vertebrate animals, in particularfrom other warm-blooded animals, more in particular from other mammals,and in particular from other species of primates, such as the baboonsused in the Examples below. However, as with anti-vWF Nanobodiesdescribed in WO 04/062551, the present invention in its broadest senseis not particularly limited to or defined by a specific epitope, domainor confirmation of vWF against which the Nanobodies and polypeptides ofthe invention are directed. However, it is generally assumed andpreferred that the Nanobodies and polypeptides of the invention aredirected against the A1 domain of vWF, either in its activated ornon-activated confirmation.

Thus, in a first aspect, the invention relates to a Nanobody (as definedherein), against vWF, which consist of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which:

-   i) CDR1 comprises or essentially consists of an amino acid sequence    chosen from the group consisting of:

NYGMG [SEQ ID NO: 15] SYTLG [SEQ ID NO: 16] NYNMG [SEQ ID NO: 17] SSAMA[SEQ ID NO: 18] YYNTG [SEQ ID NO: 19] IGAMG [SEQ ID NO: 20] IGTMG [SEQID NO: 21] YNPMG [SEQ ID NO: 22]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   ii) CDR2 comprises or essentially consist of an amino acid sequence    chosen from the group consisting of:

SISWSGTYTAYSDNVKG [SEQ ID NO: 23] GISWSGVSTDYAEFAKG [SEQ ID NO: 24]TSISWSGSYTAYADNVKG [SEQ ID NO: 25] SISWSGMSTYYTDSVKG [SEQ ID NO: 26]TITSGGRTSYADSVKG [SEQ ID NO: 27] AISWSGGLTYYADSVKG [SEQ ID NO: 28]TITSGGSTNYADPVKG [SEQ ID NO: 29] TITSGGSTNYADSVKG [SEQ ID NO: 30]AISRTGGSTYYARSVEG [SEQ ID NO: 31] AISRTGGSTYYPDSVEG [SEQ ID NO: 32]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   iii) CDR3 comprises or essentially consists of an amino acid    sequence chosen from the group consisting of:

QSRYRSNYYDHDDKYAY [SEQ ID NO: 33] LGRYRSNWRNIGQYDY [SEQ ID NO: 34]QSRYSSNYYDHDDKYAY [SEQ ID NO: 35] SNRYRTHTTQAMYNY [SEQ ID NO: 36]VVDGKRAP [SEQ ID NO: 37] NRRQKTVQMGERAYDY [SEQ ID NO: 38] NLKQGSYGYRFNDY[SEQ ID NO: 39] NLKQGDYGYRFNDY [SEQ ID NO: 40] AGVRAEDGRVRTLPSEYNF [SEQID NO: 41] AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42] AGVRAEDGRVRSLPSEYTF [SEQID NO: 43]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s).

In another aspect, the invention relates to a Nanobody (as definedherein), against vWF, which consist of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which:

-   i) CDR1 is an amino acid sequence chosen from the group consisting    of:

NYGMG [SEQ ID NO: 15] SYTLG [SEQ ID NO: 16] NYNMG [SEQ ID NO: 17] SSAMA[SEQ ID NO: 18] YYNTG [SEQ ID NO: 19] IGAMG [SEQ ID NO: 20] IGTMG [SEQID NO: 21] YNPMG [SEQ ID NO: 22]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   ii) CDR2 is an amino acid sequence chosen from the group consisting    of:

SISWSGTYTAYSDNVKG [SEQ ID NO: 23] GISWSGVSTDYAEFAKG [SEQ ID NO: 24]TSISWSGSYTAYADNVKG [SEQ ID NO: 25] SISWSGMSTYYTDSVKG [SEQ ID NO: 26]TITSGGRTSYADSVKG [SEQ ID NO: 27] AISWSGGLTYYADSVKG [SEQ ID NO: 28]TITSGGSTNYADPVKG [SEQ ID NO: 29] TITSGGSTNYADSVKG [SEQ ID NO: 30]AISRTGGSTYYARSVEG [SEQ ID NO: 31] AISRTGGSTYYPDSVEG [SEQ ID NO: 32]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   iii) CDR3 is an amino acid sequence chosen from the group consisting    of:

QSRYRSNYYDHDDKYAY [SEQ ID NO: 33] LGRYRSNWRNIGQYDY [SEQ ID NO: 34]QSRYSSNYYDHDDKYAY [SEQ ID NO: 35] SNRYRTHTTQAMYNY [SEQ ID NO: 36]VVDGKRAP [SEQ ID NO: 37] NRRQKTVQMGERAYDY [SEQ ID NO: 38] NLKQGSYGYRFNDY[SEQ ID NO: 39] NLKQGDYGYRFNDY [SEQ ID NO: 40] AGVRAEDGRVRTLPSEYNF [SEQID NO: 41] AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42] AGVRAEDGRVRSLPSEYTF [SEQID NO: 43]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s).

The Nanobodies against vWF as described above and as further describedhereinbelow are also referred to herein as Nanobodies of the invention.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

In another aspect, the invention relates to a Nanobody against vWF,which consist of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), whichis chosen from the group consisting of Nanobodies with the one of thefollowing combinations of CDR1, CDR2 and CDR3, respectively:

CDR1: NYGMG [SEQ ID NO: 15]; CDR2: SISWSGTYTAYSDNVKG [SEQ ID NO: 23];CDR3: QSRYRSNYYDHDDKYAY [SEQ ID NO: 33] CDR1: SYTLG [SEQ ID NO: 16];CDR2: GISWSGVSTDYAEFAKG [SEQ ID NO: 24]; CDR3: LGRYRSNWRNIGQYDY [SEQ IDNO: 34] CDR1: NYGMG [SEQ ID NO: 15]; CDR2: TSISWSGSYTAYADNVKG [SEQ IDNO: 25]; CDR3: QSRYSSNYYDHDDKYAY [SEQ ID NO: 35] CDR1: NYNMG [SEQ ID NO:17]; CDR2: SISWSGMSTYYTDSVKG [SEQ ID NO: 26]; CDR3: SNRYRTHTTQAMYNY [SEQID NO: 36] CDR1: SSAMA [SEQ ID NO: 18]; CDR2: TITSGGRTSYADSVKG [SEQ IDNO: 27]; CDR3: VVDGKRAP [SEQ ID NO: 37] CDR1: YYNTG [SEQ ID NO: 19];CDR2: AISWSGGLTYYADSVKG [SEQ ID NO: 28]; CDR3: NRRQKTVQMGERAYDY [SEQ IDNO: 38] CDR1: IGAMG [SEQ ID NO: 20]; CDR2: TITSGGSTNYADPVKG [SEQ ID NO:29]; CDR3: NLKQGSYGYRFNDY [SEQ ID NO: 39] CDR1: IGAMG [SEQ ID NO: 20];CDR2: TITSGGSTNYADSVKG [SEQ ID NO: 30]; CDR3: NLKQGSYGYRFNDY [SEQ ID NO:39] CDR1: IGAMG [SEQ ID NO: 20]; CDR2: TITSGGSTNYADSVKG [SEQ ID NO: 30];CDR3: NLKQGDYGYRFNDY [SEQ ID NO: 40] CDR1: IGTMG [SEQ ID NO: 21]; CDR2:TITSGGSTNYADSVKG [SEQ ID NO: 30]; CDR3: NLKQGDYGYRFNDY [SEQ ID NO: 40]CDR1: YNPMG [SEQ ID NO: 22]; CDR2: AISRTGGSTYYARSVEG [SEQ ID NO: 31];CDR3: AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 41] CDR1: YNPMG [SEQ ID NO: 22];CDR2: AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; CDR3: AGVRAEDGRVRTLPSEYTF [SEQID NO: 42] CDR1: YNPMG [SEQ ID NO: 22]; CDR2: AISRTGGSTYYPDSVEG [SEQ IDNO: 32]; CDR3: AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]

In the Nanobodies of the invention that comprise the combinations ofCDR's mentioned above, each CDR can be replaced by a CDR chosen from thegroup consisting of amino acid sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with thementioned CDR's; in which

-   -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s);        and/or chosen from the group consisting of amino acid sequences        that have 3, 2 or only 1 (as indicated in the preceding        paragraph) “amino acid difference(s)” (as defined herein) with        the mentioned CDR(s) one of the above amino acid sequences, in        which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s).

However, of the Nanobodies of the invention that comprise thecombinations of CDR's mentioned above, Nanobodies comprising one or moreof the CDR's listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's listed above are more particularlypreferred; and Nanobodies comprising three of the CDR's listed above aremost particularly preferred.

TABLE I Preferred combinations of CDR's, of CDR's and frameworksequences, and of CDR's and humanized FR's CLONE ID FR1 ID CDR1 FR2 IDCDR1 ID FR3 CDR3 ID FR4 12A5 122 AVQLVESGGG 148 IGAMG 174 MYRQAPGK 200TITSGGSTNY 226 RFTISRDGPKNT 252 NLKQGSYGYRFN 278 WGQGTQVT LVQPGGSLRLQRELVA ADPVKG VYLQMNSLKPED DY VSS SCLASGRIFS TAVYYCYA 12B1 123QVQLVESGGG 149 NYGMG 175 WFRQAPGK 201 SISWSGTYT 227 RFTISRDNAKNT 253QSRYRSNYYDHD 279 WGQGTQVT LVQAGGSLRL EREFVT AYSDNVKG VYLQMDSLKPED DKYAYVSS SCAASGRTFS TAVYYCAA 12B6 124 QVQLVESGGG 150 YNPMG 176 WFRQAPGK 202AISRTGGST 228 RFTISRDNAKRM 254 AGVRAEDGRVRT 280 WGQGTQVT LVQAGGALRLERDVVA YYARSVEG VYLQMNALKPED LPSEYNF VSS SCAASGRTFS TAVYYCAA 12D11 125AVQLVDSGGG 151 SSYTLG 177 WFRQAPGK 203 GISWSGVST 229 RFTISRDHAANT 255LGRYRSNWRNIG 281 WGQGTQVT LVQAGGSLRL EREFVG DYAEFAKG VYLEMNSLKPED QYDYVSS SCTASERTTF TAVYYCAA 12-E3 126 EVQLVESGGG 152 NYGMG 178 WFRQAPGK 204SISWSGSYT 230 RFTISRDNAKNT 256 QSRYSSNYYDHD 282 WGQGTQVT LVQAGGSLRLEREFVT AYADNVKG VYLQMDSLKPGD DKYAY VSS SCAASGRTFN TAVYYCAA 12C9 127AVQLVESGGG 153 SSAMA 179 WYRQASGK 205 TITSGGRTSY 231 RFTISRDNAKNT 257VVDGKRAP 283 WGQGTQVT LVQPGGSLKL QRELVA ADSVKG VYLQMNSLKPED VSSSCATSGSIFS TAVYDCNF 14F8 128 AVQLVESGGG 154 YYNTG 180 WFRQAPGK 206AISWSGGLT 232 RFTISRDNAKDM 258 NRRQKTVQMGER 284 WGQGTQVT LVQAGESLRLEREFVA YYADSVKG VYLQMASLKPED AYD VSS SCTSSGRAFS TAVYYCAA 12B4 129QVQLVESGGG 155 IGAMG 181 LYRQAPGK 207 TITSGGSTNY 233 RFTISRDGPKNT 259NLKQGSYGYRFN 285 WGQGTQVT LVQPGGSLRL QRELVA ADSVKG VYLQMNSLKPED DY VSSSCLASGRIFS TAVYYCYA 12-E8 130 AVQLEESGGG 156 IGAMG 182 LYRQAPGK 208TITSGGSTNY 234 RFTISRDGAKNT 260 NLKQGDYGYRFN 286 WGQGTQVT LVQPGGSLRLQRELVA ADSVKG VYLQMNSLKPED DY VSS SCLASGRIFS TAVYYCYA 12A6 131QVQLVESGGG 157 IGTMG 183 LYRQAPGK 209 TITSGGSTNY 235 RFTISRDGAKNT 261NLKQGDYGYRFN 287 WGQGTQVT LVQPGGSLRL QRELVA ADSVKG VYLQMNSLRPED DY VSSSCLASGRIFS TAVYYCYA 12D8 132 AVQLVESGGG 158 IGTMG 184 LYRQAPGK 210TITSGGSTNY 236 RFTISRDGAKNT 262 NLKQGDYGYRFN 288 WGQGTQVT LVQPGGSLRLQRELVA ADSVKG VYLQMNSLRPED DY VSS SCLASGRIFS TAVYYCYA 12A2 133QVKLEESGGG 159 YNPMG 185 WFRQAPGK 211 AISRTGGST 237 RFTISRDNAKRM 263AGVRAEDGRVRT 289 WGQGTQVT LVQAGGALRL ERDLVA YYPDSVEG VYLQMNNLKPEDLPSEYTF VSS SCAASGRTFS TAVYYCAA 12F2 134 QVKLVESGGG 160 YNPMG 186WFRQAPGR 212 AISRTGGST 238 RFTISRDNAKRM 264 AGVRAEDGRVRS 290 WGQGTQVTLVQAGGALRL ERDVVA YYPDSVEG VYLQMNNLKPED LPSEYTF VSS SCAASGRTFS TAVYYCAA14H10 135 QVKLEESGGG 161 YNPMG 187 WFRQAPGK 213 AISRTGGST 239RFTISRDNAKRM 265 AGVRAEDGRVRT 291 WGQGTQVT LVQAGGALRL ERDVVA YYPDSVEGVYLEMNNLKPDD LPSEYTF VSS SCAASGRTFS TAVYYCAA 12B6H1 136 EVQLVESGGG 162YNPMG 188 WFRQAPGK 214 AISRTGGST 240 RFTISRDNAKRM 266 AGVRAEDGRVRT 292WGQGTQVT LVQPGGSLRL GRDVVA YYARSVEG VYLQMNSLRAED LPSEYNF VSS SCAASGRTFSTAVYYCAA 12B6H2 137 EVQLVESGGG 163 YNPMG 189 WFRQAPGK 215 AISRTGGST 241RFTISRDNAKRM 267 AGVRAEDGRVRT 293 WGQGTQVT LVQPGGSLRL GREVVA YYARSVEGVYLQMNSLRAED LPSEYNF VSS SCAASGRTFS TAVYYCAA 12B6H3 138 EVQLVESGGG 164YNPMG 190 WFRQAPGK 216 AISRTGGST 242 RFTISRDNAKNM 268 AGVRAEDGRVRT 294WGQGTQVT LVQPGGSLRL GRDVVA YYARSVEG VYLQMNSLRAED LPSEYNF VSS SCAASGRTFSTAVYYCAA 12B6H4 139 EVQLVESGGG 165 YNPMG 191 WFRQAPGK 217 AISRTGGST 243RFTISRDNAKRS 269 AGVRAEDGRVRT 295 WGQGTQVT LVQPGGSLRL GRDVVA YYARSVEGVYLQMNSLRAED LPSEYNF VSS SCAASGRTFS TAVYYCAA 12A2H1 140 EVQLVESGGG 166YNPMG 192 WFRQAPGK 218 AISRTGGST 244 RFTISRDNAKRM 270 AGVRAEDGRVRT 296WGQGTQVT LVQPGGSLRL GRELVA YYPDSVEG VYLQMNSLRAED LPSEYTF VSS SCAASGRTFSTAVYYCAA 12A2H3 141 EVQLVESGGG 167 YNPMG 193 WFRQAPGK 219 AISRTGGST 245RFTISRDNAKNM 271 AGVRAEDGRVRT 297 WGQGTQVT LVQPGGSLRL GRELVA YYPDSVEGVYLQMNSLRAED LPSEYTF VSS SCAASGRTFS TAVYYCAA 12A2H4 142 EVQLVESGGG 168YNPMG 194 WFRQAPGK 220 AISRTGGST 246 RFTISRDNAKRS 272 AGVRAEDGRVRT 298WGQGTQVT LVQPGGSLRL GRELVA YYPDSVEG VYLQMNSLRAED LPSEYTF VSS SCAASGRTFSTAVYYCAA 12A2H11 143 EVQLVESGGG 169 YNPMG 195 WFRQAPGK 221 AISRTGGST 247RFTISRDNAKRM 273 AGVRAEDGRVRT 299 WGQGTQVT LVQPGGSLRL GRELVA YYPDSVEGVYLQMNSLRAED LPSEYTF VSS SCAASGFTFS TAVYYCAA 12A2H13 144 EVQLVESGGG 170YNPMG 196 WFRQAPGK 222 AISRTGGST 248 RFTISRDNAKNS 274 AGVRAEDGRVRT 300WGQGTLVT LVQPGGSLRL GRELVA YYPDSVEG VYLQMNSLRAED LPSEYTF VSS SCAASGFTFSTAVYYCAA 12A5H1 145 EVQLVESGGG 171 IGAMG 197 MYRQAPGK 223 TITSGGSTNY 249RFTISRDGPKNT 275 NLKQGSYGYRFN 301 WGQGTQVT LVQPGGSLRL GRELVA ADPVKGVYLQMNSLRAED DY VSS SCAASGRIFS TAVYYCYA 12A5H2 146 EVQLVESGGG 172 IGAMG198 MYRQAPGK 224 TITSGGSTNY 250 RFTISRDGAKNT 276 NLKQGSYGYRFN 302WGQGTQVT LVQPGGSLRL GRELVA ADPVKG VYLQMNSLRAED DY VSS SCAASGRIFSTAVYYCYA 12A5H3 147 EVQLVESGGG 173 IGAMG 199 MYRQAPGK 225 TITSGGSTNY 251RFTISRDNAKNT 277 NLKQGSYGYRFN 303 WGQGTQVT LVQPGGSLRL GRELVA ADPVKGVYLQMNSLRAED DY VSS SCAASGRIFS TAVYYCYA

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table I;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table I; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1“amino acid difference(s)” (as defined herein) with at least one of theCDR1, CDR2 and CDR3 sequences, respectively, listed in Table I. In thiscontext, by “suitably chosen” is meant that, as applicable, a CDR1sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table I or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table I; and/or from the groupconsisting of the CDR3 sequences that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR3 sequences listed in Table I.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table I; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1“amino acid difference(s)” with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table I.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table I or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table I, respectively; and atleast one of the CDR1 and CDR2 sequences present is suitably chosen fromthe group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table I or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table I; and/or from the group consisting of the CDR1 and CDR2sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table I.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table I; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table I.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table I. Preferably, in this embodiment, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with at least one of the corresponding CDRsequences, respectively, listed in Table I; and/or from the groupconsisting of the CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with at least one of the corresponding sequences,respectively, listed in Table I.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table I. Preferably, in this embodiment, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thatthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with theCDR1 and CDR2 sequences, respectively, listed in listed in Table I;and/or from the group consisting of the CDR1 and CDR2 sequences,respectively, that have 3, 2 or only 1 amino acid difference(s) with atleast one of the CDR1 and CDR2 sequences, respectively, listed in TableI.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table I. Preferably, in this embodiment, the remaining CDRsequence present are suitably chosen from the group of CDR sequencesthat that have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the corresponding CDR sequences listed in Table I; and/orfrom the group consisting of CDR sequences that have 3, 2 or only 1amino acid difference(s) with at least one of the correspondingsequences listed in Table I.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table 1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table I. Preferably, in thisembodiment, the remaining CDR sequence present are suitably chosen fromthe group of CDR sequences that that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with at least one of the corresponding CDRsequences listed in Table I; and/or from the group consisting of CDRsequences that have 3, 2 or only 1 amino acid difference(s) with thecorresponding CDR sequences listed in Table I.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I.

Also, generally, the combinations of CDR's listed in Table I (i.e. thosementioned on the same line in Table I) are preferred. Thus, it isgenerally preferred that, when a CDR in a Nanobody of the invention is aCDR sequence mentioned in Table I or is suitably chosen from the groupof CDR sequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table I; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table I, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table I (i.e. mentionedon the same line in Table I) or are suitably chosen from the group ofCDR sequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in Table I.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table I, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table I (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table I; a CDR2 sequence that has 3, 2 or 1amino acid difference with one of the CDR2 sequences mentioned in TableI (but belonging to a different combination); and a CDR3 sequence thathas more than 80% sequence identity with one of the CDR3 sequencesmentioned in Table I (but belonging to a different combination); or (2)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table I; a CDR2 sequence, and one of theCDR3 sequences listed in Table I; or (3) a CDR1 sequence; a CDR2sequence that has more than 80% sequence identity with one of the CDR2sequence listed in Table I; and a CDR3 sequence that has 3, 2 or 1 aminoacid differences with the CDR3 sequence mentioned in Table I thatbelongs to the same combination as the CDR2 sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table I; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table I that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table I that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table I and a CDR3 sequence listed in TableI (in which the CDR2 sequence and CDR3 sequence may belong to differentcombinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table I; the CDR2 sequencelisted in Table I that belongs to the same combination; and a CDR3sequence mentioned in Table I that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table I; a CDR2 sequence that has 3,2 or 1 amino acid differences with the CDR2 sequence mentioned in TableI that belongs to the same combination; and more than 80% sequenceidentity with the CDR3 sequence listed in Table I that belongs to samedifferent combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table I, a CDR2 sequence that hasmore than 80% sequence identity with the CDR2 sequence mentioned inTable I that belongs to the same combination; and the CDR3 sequencementioned in Table I that belongs to the same.

In the most preferred in the Nanobodies of the invention, the CDR1, CDR2and CDR3 sequences present are suitably chosen from the one of thecombinations of CDR1, CDR2 and CDR3 sequences, respectively, listed inTable I.

Preferably, when a CDR sequence is suitably chosen from the group of CDRsequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity (as defined herein) with one of the CDR sequences listed inTable I; and/or when a CDR sequence is suitably chosen from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with one of the CDR sequences listed in Table I:

-   -   i) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   ii) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the CDR sequence listed in Table I.

According to a non-limiting but preferred embodiment of the invention,the CDR sequences in the Nanobodies of the invention are as definedabove and are also such that the Nanobody of the invention binds to vWFwith an dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter (M)or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter (M) or less and morepreferably 10⁻⁸ to 10⁻¹² moles/liter (M), and/or with an associationconstant (K_(A)) of at least 10⁷ M⁻¹, preferably at least 10⁸ M⁻¹, morepreferably at least 10⁹ M⁻¹, such as at least 10¹² M⁻; and in particularwith a K_(D) less than 500 nM, preferably less than 200 nM, morepreferably less than 10 nM, such as less than 500 pM. The K_(D) andK_(A) values of the Nanobody of the invention against vWF can bedetermined in a manner known per se, for example using the assaydescribed herein. More generally, the Nanobodies described hereinpreferably have a dissociation constant with respect to vWF that is asdescribed in this paragraph.

In another aspect, the invention relates to a Nanobody with an aminoacid sequence that is chosen from the group consisting of SEQ ID NO's:60 to 73 and SEQ ID NO's: 86 to 97 or from the group consisting of fromamino acid sequences that have more than 80%, preferably more than 90%,more preferably more than 95%, such as 99% or more “sequence identity”(as defined herein) with one or more of the amino acid sequences of SEQID NO's: 60 to 73 and SEQ ID NO's: 86 to 97, which amino acid sequencesmost preferably have framework sequences that are as further definedbelow under the general description of the framework sequences ofNanobodies.

According to a specific, but non-limiting embodiment, the latter aminoacid sequences have been “humanized”, as further described below.

Most preferably, the Nanobodies of the invention are chosen from thegroup consisting of SEQ ID NO's: 60 to 73 and SEQ ID NO's: 86 to 97, ofwhich the “humanized” Nanobodies of SEQ ID NO's: 86 to 97 may beparticularly preferred.

Nanobodies that are particular preferred according to the invention isNanobody 12B6 (SEQ ID NO: 62) and homologues and variants thereof, andin particular humanized variants thereof. Some particularly preferred,but non-limiting homologues and (humanized) variants are for exampleNanobodies 12A2 (SEQ ID NO: 71); 12F2 (SEQ ID NO: 72); 14H10 (SEQ ID NO:73) and humanized variants thereof, such as 12B6H1 (SEQ ID NO: 86);12B6H2 (SEQ ID NO: 87); 12B6H3 (SEQ ID NO: 88); 12B6H4 (SEQ ID NO: 89);12A2H1 (SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92);12A2H11 (SEQ ID NO: 93) and 12A2H113 (SEQ ID NO: 94).

Particularly preferred in the invention is Nanobody 12A2 (SEQ ID NO: 71)and homologues and variants thereof, and in particular humanizedvariants thereof. Some particularly preferred, but non-limitinghomologues and (humanized) variants are for example Nanobodies 12A2H1(SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92); 12A2H11(SEQ ID NO: 93) and 12A2H13 (SEQ ID NO: 94), of which Nanobody 12A2H1(SEQ ID NO: 90) is in particular preferred.

Thus, one preferred but non-limiting aspect of the invention relates toa Nanobody against Von Willebrand Factor (vWF), said Nanobody consistingof 4 framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

a) CDR1 comprises or essentially consists of:

-   -   the amino acid sequence YNPMG [SEQ ID NO: 22]; or    -   an amino acid sequences that has 2 or only 1 amino acid        difference(s) with the amino acid sequence YNPMG [SEQ ID NO:        22];

and

b) CDR2 comprises or essentially consists of:

-   -   the amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or    -   an amino acid sequences that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AISRTGGSTYYPDSVEG        [SEQ ID NO: 32];

and

c) CDR3 comprises or essentially consists of:

-   -   the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO:        42].

In particular, the invention relates to such a Nanobody, in which:

-   -   CDR1 comprises or essentially consists of the amino acid        sequence YNPMG [SEQ ID NO: 22];        or in which:    -   CDR2 comprises or essentially consists of the amino acid        sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32];        or in which    -   CDR3 comprises or essentially consists of the amino acid        sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].

For example, the invention relates to such Nanobodies, in which:

-   -   CDR1 comprises or essentially consists of the amino acid        sequence YNPMG [SEQ ID NO: 22]; and CDR3 comprises or        essentially consists of the amino acid sequence        AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42];        or in which:    -   CDR1 comprises or essentially consists of the amino acid        sequence YNPMG [SEQ ID NO: 22]; and CDR2 comprises or        essentially consists of the amino acid sequence        AISRTGGSTYYPDSVEG [SEQ ID NO: 32];        or in which:    -   CDR2 comprises or essentially consists of the amino acid        sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; and CDR3 comprises        or essentially consists of the amino acid sequence        AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]

In one aspect, the invention relates to such a Nanobody, in which CDR1comprises or essentially consists of the amino acid sequence YNPMG [SEQID NO: 22]; and CDR3 comprises or essentially consists of the amino acidsequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].

The invention also relates to humanized variants of such a Nanobody.Some preferred, but non-limiting humanizing substitutions will bedescribed herein, or will be clear to the skilled person by comparingthe corresponding non-humanized and humanized Nanobodies disclosedherein. Some particularly useful humanizing substitutions are one ormore of those present in the humanized variants of 12A2 (as will beclear to the skilled person from a comparison of the sequences of 12A2H1(SEQ ID NO: 90) with the corresponding humanized sequences of 12A2H3(SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and12A2H13 (SEQ ID NO: 94).

Another one preferred but non-limiting aspect of the invention relatesto a Nanobody against Von Willebrand Factor (vWF), said Nanobodyconsisting of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

d) CDR1 is:

-   -   the amino acid sequence YNPMG [SEQ ID NO: 22]; or    -   an amino acid sequences that has 2 or only 1 amino acid        difference(s) with the amino acid sequence YNPMG [SEQ ID NO:        22];

and

e) CDR2 is:

-   -   the amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; or    -   an amino acid sequences that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AISRTGGSTYYPDSVEG        [SEQ ID NO: 32];

and

f) CDR3 is:

-   -   the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; or    -   an amino acid sequences that has only 1 amino acid difference        with the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO:        42].

In particular, the invention relates to such a Nanobody, in which:

CDR1 is the amino acid sequence YNPMG [SEQ ID NO: 22];

or in which:

CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32];

or in which

CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].

For example, the invention relates to such Nanobodies, in which:

-   -   CDR1 is the amino acid sequence YNPMG [SEQ ID NO: 22]; and CDR3        is the amino acid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42];        or in which:    -   CDR1 is the amino acid sequence YNPMG [SEQ ID NO: 22]; and CDR2        is the amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO: 32];        or in which:    -   CDR2 is the amino acid sequence AISRTGGSTYYPDSVEG [SEQ ID NO:        32]; and CDR3 is the amino acid sequence AGVRAEDGRVRTLPSEYTF        [SEQ ID NO: 42]

In one aspect, the invention relates to such a Nanobody, in which CDR1is the amino acid sequence YNPMG [SEQ ID NO: 22]; and CDR3 is the aminoacid sequence AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42].

The invention also relates to humanized variants of such a Nanobody.Some preferred, but non-limiting humanizing substitutions will bedescribed herein, or will be clear to the skilled person by comparingthe corresponding non-humanized and humanized Nanobodies disclosedherein. Some particularly useful humanizing substitutions are one ormore of those present in the humanized variants of 12A2 (as will beclear to the skilled person from a comparison of the sequences of 12A2H1(SEQ ID NO: 90) with the corresponding humanized sequences of 12A2H3(SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and12A2H13 (SEQ ID NO: 94).

The Nanobodies described herein may be GLEW-class Nanobodies, “103 P, Ror S”-class Nanobodies or “KERE-class Nanobodies” (all as describedherein). In particular, the Nanobodies described herein may beKERE-class Nanobodies, although the invention is not limited thereto.

In another aspect, the invention relates to a Nanobody which has atleast 80%, or at least 90%, or at least 95%, or at least 99% sequenceidentity (as defined herein) with at least one of the Nanobodies fromthe group consisting of SEQ ID NO's 60-73 and SEQ ID NO's 86-97.

In particular, the invention relates to a Nanobody which has at least80%, or at least 90%, or at least 95%, or at least 99% sequence identity(as defined herein) with at least one of the Nanobodies 12B6 (SEQ ID NO:62); 12A2 (SEQ ID NO: 71); 12F2 (SEQ ID NO: 72); 14H10 (SEQ ID NO: 73);12B6H1 (SEQ ID NO: 86); 12B6H2 (SEQ ID NO: 87); 12B6H3 (SEQ ID NO: 88);12B6H4 (SEQ ID NO: 89); 12A2H1 (SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91);12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and/or 12A2H13 (SEQ IDNO: 94).

More in particular, the invention relates to a Nanobody which has atleast 80%, or at least 90%, or at least 95%, or at least 99% sequenceidentity (as defined herein) with at least one of the Nanobodies 12A2(SEQ ID NO: 71); 12A2H1 (SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4(SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and/or 12A2H113 (SEQ ID NO:94).

Even more in particular, the invention relates to a Nanobody which hasat least 80%, or at least 90%, or at least 95%, or at least 99% sequenceidentity (as defined herein) with the Nanobody 12A2H1 (SEQ ID NO: 90).

The invention also relates to humanized variants of such Nanobodies.Some preferred, but non-limiting humanizing substitutions will bedescribed herein, or will be clear to the skilled person by comparingthe corresponding non-humanized and humanized Nanobodies disclosedherein. Some particularly useful humanizing substitutions are one ormore of those present in the humanized variants of 12A2 (as will beclear to the skilled person from a comparison of the sequences of 12A2H1(SEQ ID NO: 90) with the corresponding humanized sequences of 12A2H3(SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92); 12A2H11 (SEQ ID NO: 93) and12A2H13 (SEQ ID NO: 94).

The invention also relates to a Nanobody that is chosen from the groupconsisting of the Nanobodies of SEQ ID NO's 60-73 and SEQ ID NO's 86-97.

In particular, the invention relates to a Nanobody that is chosen fromthe group consisting of the Nanobodies 12B6 (SEQ ID NO: 62); 12A2 (SEQID NO: 71); 12F2 (SEQ ID NO: 72); 14H10 (SEQ ID NO: 73); 12B6H1 (SEQ IDNO: 86); 12B6H2 (SEQ ID NO: 87); 12B6H3 (SEQ ID NO: 88); 12B6H4 (SEQ IDNO: 89); 12A2H1 (SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ IDNO: 92); 12A2H11 (SEQ ID NO: 93) and/or 12A2H13 (SEQ ID NO: 94).

More in particular, the invention relates to a Nanobody that is chosenfrom the group consisting of the Nanobodies 12A2 (SEQ ID NO: 71); 12A2H1(SEQ ID NO: 90); 12A2H3 (SEQ ID NO: 91); 12A2H4 (SEQ ID NO: 92); 12A2H11(SEQ ID NO: 93) and/or 12A2H13 (SEQ ID NO: 94). A particularly usefulNanobody is Nanobody 12A2H1 (SEQ ID NO:90).

The Nanobodies described herein preferably have framework sequences thatare as further described herein. Some particularly preferred frameworksequences (FR1, FR2, FR3 and FR4, respectively) are those of Nanobody12A2 and its humanized variants; and framework sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withone of said framework sequences; and/or from the group consisting ofamino acid sequences that have 3, 2 or only 1 “amino acid difference(s)”(as defined herein) with one of said framework sequences (in which anyamino acid substitution is preferably a conservative amino acidsubstitution; and/or in which said amino acid sequence preferablycontains amino acid substitutions and no more than 3 amino aciddeletions or no more than 3 amino acid insertions). Nanobodies againstvWF with such framework sequences form a further aspect of theinvention.

In particular, the invention relates to a Nanobody against vWF, in whichFR1 is SEQ ID NO: 140; FR2 is SEQ ID NO: 192; FR3 is SEQ ID 244; and FR4is SEQ ID NO: 296; or framework sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with oneof said framework sequences; and/or from the group consisting of aminoacid sequences that have 3, 2 or only 1 “amino acid difference(s)” (asdefined herein) with one of said framework sequences (in which any aminoacid substitution is preferably a conservative amino acid substitution;and/or in which said amino acid sequence preferably contains amino acidsubstitutions and no more than 3 amino acid deletions or no more than 3amino acid insertions).

More in particular, the invention relates to a Nanobody against vWF, inwhich FR1 is SEQ ID NO: 140; FR2 is SEQ ID NO: 192; FR3 is SEQ ID 244;and FR4 is SEQ ID NO: 296.

In another aspect, the invention relates to a polypeptide that comprisesor essentially consists of at least one Nanobody against vWF as definedherein. Such polypeptides are also referred to herein as “polypeptidesof the invention” and may be as further described hereinbelow and/or asgenerally described in WO 02/062551 for the Nanobodies disclosedtherein, and may for example be multivalent polypeptides ormultispecific polypeptides, again as further described hereinbelow.

Preferably, a polypeptide of the invention is either bivalent ortrivalent (i.e. comprising two or three Nanobodies of the invention,respectively, optionally linked via one or two linkers as definedherein, respectively) or a multispecific polypeptide, comprising one ortwo, and preferably two, Nanobodies of the invention and at least oneNanobody directed against a serum protein, and in particular against ahuman serum protein, such as against human serum albumin.

In one preferred, but non-limiting embodiments, the Nanobodies of theinvention present in the polypeptides of the invention are chosen fromthe group consisting of SEQ ID NO's: 60 to 73 and SEQ ID NO's: 86 to 97,and in particular from the “humanized” Nanobodies of SEQ ID NO's 86 to97. The Nanobodies against human serum albumin present in thepolypeptides of the invention are preferably as defined herein, and aremore preferably chosen from the group consisting of SEQ ID NO's: 107 to121, and in particular from the “humanized” Nanobodies against humanserum albumin of SEQ ID NO's 114-121.

Some preferred, but non-limiting examples of polypeptides of theinvention are the polypeptides of SEQ ID NO's: 74 to 82 and thepolypeptides of SEQ ID NO's 98-106. Other polypeptides of the inventionmay for example be chosen from the group consisting of amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the amino acid sequences of SEQ IDNO's: 74 to 82 and/or SEQ ID NO's 98 to 106, in which the Nanobodiescomprised within said amino acid sequences are preferably as definedherein.

According to one aspect of the invention, the Nanobodies, proteins andpolypeptides described herein have essentially no influence on thecleavage of ULvWF by ADAMTS-13. In particular, when the Nanobodies,proteins and polypeptides described herein are used at the dosesdescribed herein, the cleavage of ULvWF by ADAMTS-13 (either in vivoupon administration and/or as measured using a suitable assay, such asthe assay described herein), essentially does not reduce or inhibit thecleavage of ULvWF by ADAMTS-13, i.e. by not more than 50%, preferablynot more than 20%, even more preferably not more than 10%, such as lessthan 5% or essentially not at all). Thus, one further aspect of theinvention relates to a Nanobody, protein or polypeptide, and inparticular a Nanobody, protein or polypeptide as described herein, thatessentially does not reduce or inhibit the cleavage of ULvWF byADAMTS-13.

In another aspect, the invention relates to a nucleic acid that encodesa Nanobody of the invention and/or a polypeptide of the invention. Sucha nucleic acid will also be referred to below as a “nucleic acid of theinvention” and may for example be in the form of a genetic construct, asdefined herein.

In another aspect, the invention relates to host or host cell thatexpresses or is capable of expressing a Nanobody of the invention and/ora polypeptide of the invention; and/or that contains a nucleic acidencoding a Nanobody of the invention and/or a polypeptide of theinvention. Such a host or a host cell may also be analogous to the hostsand host cells described in WO 02/062551, but expressing or capable ofexpressing a Nanobody of the invention and/or a polypeptide of theinvention and/or containing a nucleic acid as described herein.

The invention further relates to a product or composition containing orcomprising a Nanobody of the invention, a polypeptide of the invention;and/or a nucleic acid of the invention. Such a product or compositionmay for example be a pharmaceutical composition (as described below) ora product or composition for diagnostic use (as also described below).Such a product or composition may also be analogous to the products andcompositions described in WO 02/062551, but containing or comprising aNanobody of the invention, a polypeptide of the invention or a nucleicacid of the invention.

The invention further relates to methods for preparing or generating theNanobodies, polypeptides, nucleic acids, host cells, products andcompositions as described herein, which methods are as further describedbelow. Also, generally, the Nanobodies, polypeptides, nucleic acids,host cells, products and compositions described herein may also beprepared and used in a manner analogous to the manner described in WO02/062551.

The invention further relates to applications and uses of the aboveNanobodies, polypeptides, nucleic acids, host cells, products andcompositions described herein, which applications and uses include, butare not limited to, the applications and uses described hereinbelowand/or the further uses and applications for Nanobodies against vWFand/or for polypeptides containing the same in WO 02/062551.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION

The above and other aspects and embodiments of the invention will becomeclear from the further description hereinbelow, in which:

a) Unless indicated or defined otherwise, all terms used have theirusual meaning in the art, which will be clear to the skilled person.Reference is for example made to the standard handbooks, such asSambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.),Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al,eds., “Current protocols in molecular biology”, Green Publishing andWiley Interscience, New York (1987); Roitt et al., “Immunology” (6th.Ed.), Mosby/Elsevier, Edinburgh (2001); and Janeway et al.,“Immunobiology” (6^(th) Ed.), Garland Science Publishing/ChurchillLivingstone, New York (2005), as well as to the general background artcited above;b) Unless indicated otherwise, the term “immunoglobulinsequence”—whether it used herein to refer to a heavy chain antibody orto a conventional 4-chain antibody—is used as a general term to includeboth the full-size antibody, the individual chains thereof, as well asall parts, domains or fragments thereof (including but not limited toantigen-binding domains or fragments such as V_(HH) domains orV_(H)/V_(L) domains, respectively). In addition, the term “sequence” asused herein (for example in terms like “immunoglobulin sequence”,“antibody sequence”, “variable domain sequence”, “V_(HH) sequence” or“protein sequence”), should generally be understood to include both therelevant amino acid sequence as well as nucleic acid sequences ornucleotide sequences encoding the same, unless the context requires amore limited interpretation;c) Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks, to the general background art referred to above andto the further references cited therein;d) Amino acid residues will be indicated according to the standardthree-letter or one-letter amino acid code, as mentioned in Table 1;

TABLE 1 one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residu can generallybe considered essentially uncharged at a pH of about 6.5.e) For the purposes of comparing two or more nucleotide sequences, thepercentage of “sequence identity” between a first nucleotide sequenceand a second nucleotide sequence may be calculated by dividing [thenumber of nucleotides in the first nucleotide sequence that areidentical to the nucleotides at the corresponding positions in thesecond nucleotide sequence] by [the total number of nucleotides in thefirst nucleotide sequence] and multiplying by [100%], in which eachdeletion, insertion, substitution or addition of a nucleotide in thesecond nucleotide sequence—compared to the first nucleotide sequence—isconsidered as a difference at a single nucleotide (position).Alternatively, the degree of sequence identity between two or morenucleotide sequences may be calculated using a known computer algorithmfor sequence alignment such as NCBI Blast v2.0, using standard settings.

Some other techniques, computer algorithms and settings for determiningthe degree of sequence identity are for example described in WO04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO98/49185 and GB 2 357 768-A.

Usually, for the purpose of determining the percentage of “sequenceidentity” between two nucleotide sequences in accordance with thecalculation method outlined hereinabove, the nucleotide sequence withthe greatest number of nucleotides will be taken as the “first”nucleotide sequence, and the other nucleotide sequence will be taken asthe “second” nucleotide sequence;

f) For the purposes of comparing two or more amino acid sequences, thepercentage of “sequence identity” between a first amino acid sequenceand a second amino acid sequence may be calculated by dividing [thenumber of amino acid residues in the first amino acid sequence that areidentical to the amino acid residues at the corresponding positions inthe second amino acid sequence] by [the total number of nucleotides inthe first amino acid sequence] and multiplying by [100%], in which eachdeletion, insertion, substitution or addition of an amino acid residuein the second amino acid sequence—compared to the first amino acidsequence—is considered as a difference at a single amino acid residue(position), i.e. as an “amino acid difference” as defined herein.

Alternatively, the degree of sequence identity between two amino acidsequences may be calculated using a known computer algorithm, such asthose mentioned above for determining the degree of sequence identityfor nucleotide sequences, again using standard settings.

Usually, for the purpose of determining the percentage of “sequenceidentity” between two amino acid sequences in accordance with thecalculation method outlined hereinabove, the amino acid sequence withthe greatest number of amino acid residues will be taken as the “first”amino acid sequence, and the other amino acid sequence will be taken asthe “second” amino acid sequence.

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, which can generally bedescribed as amino acid substitutions in which an amino acid residue isreplaced with another amino acid residue of similar chemical structureand which has little or essentially no influence on the function,activity or other biological properties of the polypeptide. Suchconservative amino acid substitutions are well known in the art, forexample from WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 andWO 01/09300; and (preferred) types and/or combinations of suchsubstitutions may be selected on the basis of the pertinent teachingsfrom WO 04/037999 as well as WO 98/49185 and from the further referencescited therein.

Such conservative substitutions preferably are substitutions in whichone amino acid within the following groups (a)-(e) is substituted byanother amino acid residue within the same group: (a) small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b)polar, negatively charged residues and their (uncharged) amides: Asp,Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg andLys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys;and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Nad. Acad.Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J. Molec. Biol. 157:105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353,1986, all incorporated herein in their entirety by reference.Information on the primary, secondary and tertiary structure ofNanobodies given in the description below and in the general backgroundart cited above. Also, for this purpose, the crystal structure of aV_(HH) domain from a llama is for example given by Desmyter et al.,Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al.,Natural Structural Biology (1996); 3, 752-757; and Decanniere et al.,Structure, Vol. 7, 4, 361 (1999);

g) amino acid sequences and nucleic acid sequences are said to be“exactly the same” if they have 100% sequence identity (as definedherein) over their entire length;

h) when comparing two amino acid sequences, the term “amino aciddifference” refers to an insertion, deletion or substitution of a singleamino acid residue on a position of the first sequence, compared to thesecond sequence; it being understood that two amino acid sequences cancontain one, two or more such amino acid differences;i) a nucleic acid sequence or amino acid sequence is considered to be“(in) essentially isolated form)”—for example, compared to its nativebiological source and/or the reaction medium or cultivation medium fromwhich it has been obtained—when it has been separated from at least oneother component with which it is usually associated in said source ormedium, such as another nucleic acid, another protein/polypeptide,another biological component or macromolecule or at least onecontaminant, impurity or minor component. In particular, a nucleic acidsequence or amino acid sequence is considered “essentially isolated”when it has been purified at least 2-fold, in particular at least10-fold, more in particular at least 100-fold, and up to 1000-fold ormore. A nucleic acid sequence or amino acid sequence that is “inessentially isolated form” is preferably essentially homogeneous, asdetermined using a suitable technique, such as a suitablechromatographical technique, such as polyacrylamide-gelelectrophoresis;j) The term “domain” as used herein generally refers to a globularregion of an antibody chain, and in particular to a globular region of aheavy chain antibody, or to a polypeptide that essentially consists ofsuch a globular region. Usually, such a domain will comprise peptideloops (for example 3 or 4 peptide loops) stabilized, for example, as asheet or by disulfide bonds.k) The term ‘antigenic determinant’ refers to the epitope on the antigenrecognized by the antigen-binding molecule (such as a Nanobody or apolypeptide of the invention) and more in particular by theantigen-binding site of said molecule. The terms “antigenic determinant”and “epitope” may also be used interchangeably herein.l) An amino acid sequence (such as a Nanobody, an antibody, apolypeptide of the invention, or generally an antigen binding protein orpolypeptide or a fragment thereof) that can bind to, that has affinityfor and/or that has specificity for a specific antigenic determinant,epitope, antigen or protein (or for at least one part, fragment orepitope thereof) is said to be “against” or “directed against” saidantigenic determinant, epitope, antigen or protein.m) The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein (such as a Nanobody or a polypeptideof the invention) molecule can bind. The specificity of anantigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an antigenicdeterminant and the antigen-binding molecule (alternatively, theaffinity can also be expressed as the affinity constant (KA), which is1/KD). As will be clear to the skilled person (for example on the basisof the further disclosure herein), affinity can be determined in amanner known per se, depending on the specific antigen of interest.Avidity is the measure of the strength of binding between anantigen-binding molecule (such as a Nanobody or polypeptide of theinvention) and the pertinent antigen. Avidity is related to both theaffinity between an antigenic determinant and its antigen binding siteon the antigen-binding molecule and the number of pertinent bindingsites present on the antigen-binding molecule. Typically,antigen-binding proteins (such as the Nanobodies and/or polypeptides ofthe invention) will bind with a dissociation constant (KD) of 10⁻⁵ to10⁻¹² moles/liter (M) or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter(M) or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter, and/or withan association constant (K_(A)) of at least 10⁷ M⁻¹, preferably at least10⁸ M⁻¹, more preferably at least 10⁹ M-1, such as at least 10¹² M⁻¹.Any K_(D) value greater than 10⁻⁴ M is generally considered to indicatenon-specific binding. Preferably, a Nanobody or polypeptide of theinvention will bind to the desired antigen with an K_(D) less than 500nM, preferably less than 200 nM, more preferably less than 10 nM, suchas less than 500 pM. Specific binding of an antigen-binding protein toan antigen or antigenic determinant can be determined in any suitablemanner known per se, including, for example, Scatchard analysis and/orcompetitive binding assays, such as radioimmunoassays (RIA), enzymeimmunoassays (EIA) and sandwich competition assays, and the differentvariants thereof known per se in the art.n) as further described hereinbelow, the amino acid sequence andstructure of a Nanobody can be considered—without however being limitedthereto—to be comprised of four framework regions or “FR's”, which arereferred to in the art and hereinbelow as “Framework region 1” or “FR1”;as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; andas “Framework region 4” or “FR4”, respectively; which framework regionsare interrupted by three complementary determining regions or “CDR's”,which are referred to in the art as “Complementarity Determining Region1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; andas “Complementarity Determining Region 3” or “CDR3”, respectively;o) as also further describe hereinbelow, the total number of amino acidresidues in a Nanobody can be in the region of 110-120, is preferably112-115, and is most preferably 113. It should however be noted thatparts, fragments or analogs (as further described hereinbelow) of aNanobody are not particularly limited as to their length and/or size, aslong as such parts, fragments or analogs meet the further requirementsoutlined hereinbelow and are also preferably suitable for the purposesdescribed herein;p) the amino acid residues of a Nanobody are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to V_(HH) domains fromCamelids in the article of Riechmann and Muyldermans, referred to above(see for example FIG. 2 of said reference). According to this numbering,FR1 of a Nanobody comprises the amino acid residues at positions 1-30,CDR1 of a Nanobody comprises the amino acid residues at positions 31-36,FR2 of a Nanobody comprises the amino acids at positions 36-49, CDR2 ofa Nanobody comprises the amino acid residues at positions 50-65, FR3 ofa Nanobody comprises the amino acid residues at positions 66-94, CDR3 ofa Nanobody comprises the amino acid residues at positions 95-102, andFR4 of a Nanobody comprises the amino acid residues at positions103-113. [In this respect, it should be noted that—as is well known inthe art for V_(H) domains and for V_(HH) domains—the total number ofamino acid residues in each of the CDR's may vary and may not correspondto the total number of amino acid residues indicated by the Kabatnumbering (that is, one or more positions according to the Kabatnumbering may not be occupied in the actual sequence, or the actualsequence may contain more amino acid residues than the number allowedfor by the Kabat numbering). This means that, generally, the numberingaccording to Kabat may or may not correspond to the actual numbering ofthe amino acid residues in the actual sequence. Generally, however, itcan be said that, according to the numbering of Kabat and irrespectiveof the number of amino acid residues in the CDR's, position 1 accordingto the Kabat numbering corresponds to the start of FR1 and visa versa,position 36 according to the Kabat numbering corresponds to the start ofFR2 and visa versa, position 66 according to the Kabat numberingcorresponds to the start of FR3 and visa versa, and position 103according to the Kabat numbering corresponds to the start of FR4 andvisa versa.].

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner toV_(HH) domains from Camelids and to Nanobodies, are the method describedby Chothia et al. (Nature 342, 877-883 (1989)), the so-called “AbMdefinition” and the so-called “contact definition”. However, in thepresent description, claims and figures, the numbering according toKabat as applied to V_(HH) domains by Riechmann and Muyldermans will befollowed, unless indicated otherwise; and

q) the Figures, Sequence Listing and the Experimental Part/Examples areonly given to further illustrate the invention and should not beinterpreted or construed as limiting the scope of the invention and/orof the appended claims in any way, unless explicitly indicated otherwiseherein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the followingreferences, which are mentioned as general background art: WO 94/04678,WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and applicant; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551 by applicant and the further published patentapplications by applicant;

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As mentioned above, the invention generally relates to Nanobodiesdirected against vWF, as well as to polypeptides comprising oressentially consisting of one or more of such Nanobodies, that can beused for the prophylactic, therapeutic and/or diagnostic purposesdescribed below and in WO 04/062551.

As also mentioned above and further described below, the inventionfurther relates to nucleic acids encoding such Nanobodies andpolypeptides, to methods for preparing such Nanobodies and polypeptides,to host cells expressing or capable of expressing such Nanobodies orpolypeptides, to uses of such Nanobodies, polypeptides, nucleic acids orhost cells, and to compositions comprising such Nanobodies,polypeptides, nucleic acids or host cells.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can be obtained (1)by isolating the V_(HH) domain of a naturally occurring heavy chainantibody; (2) by expression of a nucleotide sequence encoding anaturally occurring V_(HH) domain; (3) by “humanization” (as describedbelow) of a naturally occurring V_(HH) domain or by expression of anucleic acid encoding a such humanized V_(HH) domain; (4) by“camelization” (as described below) of a naturally occurring V_(H)domain from any animal species, in particular a species of mammal, suchas from a human being, or by expression of a nucleic acid encoding sucha camelized V_(H) domain; (5) by “camelisation” of a “domain antibody”or “Dab” as described by Ward et al (supra), or by expression of anucleic acid encoding such a camelized V_(H) domain; (6) using syntheticor semi-synthetic techniques for preparing proteins, polypeptides orother amino acid sequences; (7) by preparing a nucleic acid encoding aNanobody using techniques for nucleic acid synthesis, followed byexpression of the nucleic acid thus obtained; and/or (8) by anycombination of the foregoing. Suitable methods and techniques forperforming the foregoing will be clear to the skilled person based onthe disclosure herein and for example include the methods and techniquesdescribed in more detail hereinbelow.

However, according to a specific embodiment, the Nanobodies of theinvention do not have an amino acid sequence that is exactly the same as(i.e. as a degree of sequence identity of 100% with) the amino acidsequence of a naturally occurring V_(H) domain, such as the amino acidsequence of a naturally occurring V_(H) domain from a mammal, and inparticular from a human being.

One particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(HH) domain, but that hasbeen “humanized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of said naturally occurring V_(HH) sequence byone or more of the amino acid residues that occur at the correspondingposition(s) in a V_(H) domain from a conventional 4-chain antibody froma human being (e.g. indicated above). This can be performed in a mannerknown per se, which will be clear to the skilled person, for example onthe basis of the further description below and the prior art onhumanization referred to herein. Again, it should be noted that suchhumanized Nanobodies of the invention can be obtained in any suitablemanner known per se (i.e. as indicated under points (1)-(8) above) andthus are not strictly limited to polypeptides that have been obtainedusing a polypeptide that comprises a naturally occurring V_(HH) domainas a starting material.

Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain that has been“camelized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description below. Reference is also made to WO 94/04678.Such camelization may preferentially occur at amino acid positions whichare present at the V_(H)-V_(L) interface and at the so-called Camelidaehallmark residues (see for example also WO 94/04678), as also mentionedbelow. Preferably, the V_(H) domain or sequence that is used as astarting material or starting point for generating or designing thecamelized Nanobody is preferably a V_(H) sequence from a mammal, morepreferably the V_(H) sequence of a human being. However, it should benoted that such camelized Nanobodies of the invention can be obtained inany suitable manner known per se (i.e. as indicated under points (1)-(8)above) and thus are not strictly limited to polypeptides that have beenobtained using a polypeptide that comprises a naturally occurring V_(H)domain as a starting material.

For example, again as further described below, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes such a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence such that the new nucleotide sequenceencodes a humanized or camelized Nanobody of the invention,respectively, and then expressing the nucleotide sequence thus obtainedin a manner known per se so as to provide the desired Nanobody of theinvention. Alternatively, based on the amino acid sequence of anaturally occurring V_(HH) domain or V_(H) domain, respectively, theamino acid sequence of the desired humanized or camelized Nanobody ofthe invention, respectively, can be designed and then synthesized denovo using techniques for peptide synthesis known per se. Also, based onthe amino acid sequence or nucleotide sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, a nucleotide sequenceencoding the desired humanized or camelized Nanobody of the invention,respectively, can be designed and then synthesized de novo usingtechniques for nucleic acid synthesis known per se, after which thenucleotide sequence thus obtained can be expressed in a manner known perse so as to provide the desired Nanobody of the invention.

Other suitable ways and techniques for obtaining Nanobodies of theinvention and/or nucleotide sequences and/or nucleic acids encoding thesame, starting from (the amino acid sequence of) naturally occurringV_(H) domains or preferably V_(HH) domains and/or from nucleotidesequences and/or nucleic acid sequences encoding the same will be clearfrom the skilled person, and may for example comprising combining one ormore amino acid sequences and/or nucleotide sequences from naturallyoccurring V_(H) domains (such as one or more FR's and/or CDR's) with oneor more one or more amino acid sequences and/or nucleotide sequencesfrom naturally occurring V_(HH) domains (such an one or more FR's orCDR's), in a suitable manner so as to provide (a nucleotide sequence ornucleic acid encoding) a Nanobody of the invention.

According to one preferred, but non-limiting aspect of the aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which

-   i) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:

-   ii) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:

-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:

-   iv) CDR 1 is an amino acid sequence that is chosen from the group    consisting of the following amino acid sequences:

NYGMG [SEQ ID NO: 15] SYTLG [SEQ ID NO: 16] NYNMG [SEQ ID NO: 17] SSAMA[SEQ ID NO: 18] YYNTG [SEQ ID NO: 19] IGAMG [SEQ ID NO: 20] IGTMG [SEQID NO: 21] YNPMG [SEQ ID NO: 22]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   v) CDR 2 is an amino acid sequence that is chosen from the group    consisting of the following amino acid sequences:

SISWSGTYTAYSDNVKG [SEQ ID NO: 23] GISWSGVSTDYAEFAKG [SEQ ID NO: 24]TSISWSGSYTAYADNVKG [SEQ ID NO: 25] SISWSGMSTYYTDSVKG [SEQ ID NO: 26]TITSGGRTSYADSVKG [SEQ ID NO: 27] AISWSGGLTYYADSVKG [SEQ ID NO: 28]TITSGGSTNYADPVKG [SEQ ID NO: 29] TITSGGSTNYADSVKG [SEQ ID NO: 30]AISRTGCSTYYARSVEG [SEQ ID NO: 31] AISRTGGSTYYPDSVEG [SEQ ID NO: 32]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   vi) CDR 3 is an amino acid sequence that is chosen from the group    consisting of the following amino acid sequences:

QSRYRSNYYDHDDKYAY [SEQ ID NO: 33] LGRYRSNWRNIGQYDY [SEQ ID NO: 34]QSRYSSNYYDHDDKYAY [SEQ ID NO: 35] SNRYRTHTTQAMYNY [SEQ ID NO: 36]VVDGKRAP [SEQ ID NO: 37] NRRQKTVQMGERAYDY [SEQ ID NO: 38] NLKQGSYGYRFNDY[SEQ ID NO: 39] NLKQGDYGYRFNDY [SEQ ID NO: 40] AGVRAEDGRVRTLPSEYNF [SEQID NO: 41] AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42] AGVRAEDGRVRSLPSEYTF [SEQID NO: 43]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s).

Preferably, in the Nanobodies of the invention:

-   -   when CDR1 is chosen from the group consisting of (1) NYGMG [SEQ        ID NO: 15]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) SISWSGTYTAYSDNVKG [SEQ ID NO: 23]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) QSRYRSNYYDHDDKYAY [SEQ ID NO: 33]; (2) amino acid        sequences that have at least 80%, preferably at least 90%, more        preferably at least 95%, even more preferably at least 99%        sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence;    -   when CDR1 is chosen from the group consisting of (1) SYTLG [SEQ        ID NO: 16]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) GISWSGVSTDYAEFAKG [SEQ ID NO: 24]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) LGRYRSNWRNIGQYDY [SEQ ID NO: 34]; (2) amino acid        sequences that have at least 80%, preferably at least 90%, more        preferably at least 95%, even more preferably at least 99%        sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence;    -   when CDR1 is chosen from the group consisting of (1) NYGMG [SEQ        ID NO: 15]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) TSISWSGSYTAYADNVKG [SEQ ID NO: 25]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) QSRYSSNYYDHDDKYAY [SEQ ID NO: 35]; (2) amino acid        sequences that have at least 80%, preferably at least 90%, more        preferably at least 95%, even more preferably at least 99%        sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence;    -   when CDR1 is chosen from the group consisting of (1) NYNMG [SEQ        ID NO: 15]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) SISWSGMSTYYTDSVKG [SEQ ID NO: 26]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) SNRYRTHTTQAMYNY [SEQ ID NO: 36]; (2) amino acid sequences        that have at least 80%, preferably at least 90%, more preferably        at least 95%, even more preferably at least 99% sequence        identity (as defined herein) with said amino acid sequence;        and (3) amino acid sequences that have 3, 2 or only 1 “amino        acid difference(s)” (as defined herein) with said amino acid        sequence;    -   when CDR1 is chosen from the group consisting of (1) SSAMA [SEQ        ID NO: 18]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) TITSGGRTSYADSVKG [SEQ ID NO: 27]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) VVDGKRAP [SEQ ID NO: 37]; (2) amino acid sequences that        have at least 80%, preferably at least 90%, more preferably at        least 95%, even more preferably at least 99% sequence identity        (as defined herein) with said amino acid sequence; and (3) amino        acid sequences that have 2 or only 1 “amino acid difference(s)”        (as defined herein) with said amino acid sequence;    -   when CDR1 is chosen from the group consisting of (1) YYNTG [SEQ        ID NO: 19]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) AISWSGGLTYYADSVKG [SEQ ID NO: 28]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) NRRQKTVQMGERAYDY [SEQ ID NO: 38]; (2) amino acid        sequences that have at least 80%, preferably at least 90%, more        preferably at least 95%, even more preferably at least 99%        sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence;    -   when CDR1 is chosen from the group consisting of (1) IGAMG [SEQ        ID NO: 20]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) TITSGGSTNYADPVKG [SEQ ID NO: 29]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) NLKQGSYGYRFNDY [SEQ ID NO: 39]; (2) amino acid sequences        that have at least 80%, preferably at least 90%, more preferably        at least 95%, even more preferably at least 99% sequence        identity (as defined herein) with said amino acid sequence;        and (3) amino acid sequences that have 2 or only 1 “amino acid        difference(s)” (as defined herein) with said amino acid        sequence;    -   when CDR1 is chosen from the group consisting of (1) IGAMG [SEQ        ID NO: 20]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) TITSGGSTNYADSVKG [SEQ ID NO: 30]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) NLKQGSYGYRFNDY [SEQ ID NO: 39]; (2) amino acid sequences        that have at least 80%, preferably at least 90%, more preferably        at least 95%, even more preferably at least 99% sequence        identity (as defined herein) with said amino acid sequence;        and (3) amino acid sequences that have 3, 2 or only 1 “amino        acid difference(s)” (as defined herein) with said amino acid        sequence;    -   when CDR1 is chosen from the group consisting of (1) IGAMG [SEQ        ID NO: 20]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) TITSGGSTNYADSVKG [SEQ ID NO: 30]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) NLKQGDYGYRFNDY [SEQ ID NO: 40]; (2) amino acid sequences        that have at least 80%, preferably at least 90%, more preferably        at least 95%, even more preferably at least 99% sequence        identity (as defined herein) with said amino acid sequence;        and (3) amino acid sequences that have 3, 2 or only 1 “amino        acid difference(s)” (as defined herein) with said amino acid        sequence;    -   when CDR1 is chosen from the group consisting of (1) IGTMG [SEQ        ID NO: 21]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) TITSGGSTNYADSVKG [SEQ ID NO: 30]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) NLKQGDYGYRFNDY [SEQ ID NO: 40]; (2) amino acid sequences        that have at least 80%, preferably at least 90%, more preferably        at least 95%, even more preferably at least 99% sequence        identity (as defined herein) with said amino acid sequence;        and (3) amino acid sequences that have 3, 2 or only 1 “amino        acid difference(s)” (as defined herein) with said amino acid        sequence;    -   when CDR1 is chosen from the group consisting of (1) YNPMG [SEQ        ID NO: 22]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) AISRTGGSTYYARSVEG [SEQ ID NO: 31]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) AGVRAEDGRVRTLPSEYNF [SEQ ID NO: 41]; (2) amino acid        sequences that have at least 80%, preferably at least 90%, more        preferably at least 95%, even more preferably at least 99%        sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence;    -   when CDR1 is chosen from the group consisting of (1) YNPMG [SEQ        ID NO: 22]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42]; (2) amino acid        sequences that have at least 80%, preferably at least 90%, more        preferably at least 95%, even more preferably at least 99%        sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence;    -   when CDR1 is chosen from the group consisting of (1) YNPMG [SEQ        ID NO: 22]; (2) amino acid sequences that have at least 80%,        preferably at least 90%, more preferably at least 95%, even more        preferably at least 99% sequence identity (as defined herein)        with said amino acid sequence; and (3) amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with said amino acid sequence; then CDR2 is chosen from the        group consisting of (1) AISRTGGSTYYPDSVEG [SEQ ID NO: 32]; (2)        amino acid sequences that have at least 80%, preferably at least        90%, more preferably at least 95%, even more preferably at least        99% sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; and CDR3 is chosen from the group consisting        of (1) AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]; (2) amino acid        sequences that have at least 80%, preferably at least 90%, more        preferably at least 95%, even more preferably at least 99%        sequence identity (as defined herein) with said amino acid        sequence; and (3) amino acid sequences that have 3, 2 or only 1        “amino acid difference(s)” (as defined herein) with said amino        acid sequence; in which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s).

In particular, a Nanobody against vWF according to the invention mayhave the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which        i) the amino acid residue at position 108 according to the Kabat        numbering is Q; and/or in which:        ii) the amino acid residue at position 44 according to the Kabat        numbering is E and in which the amino acid residue at position        45 according to the Kabat numbering is an R;        and/or in which:        iii) the amino acid residue at position 103 according to the        Kabat numbering is chosen from the group consisting of P, R and        S, and is in particular chosen from the group consisting of R        and S;        and in which:        iv) CDR 1 is an amino acid sequence that is chosen from the        group consisting of the following amino acid sequences:

NYGMG [SEQ ID NO: 15] SYTLG [SEQ ID NO: 16] NYNMG [SEQ ID NO: 17] SSAMA[SEQ ID NO: 18] YYNTG [SEQ ID NO: 19] IGAMG [SEQ ID NO: 20] IGTMG [SEQID NO: 21] YNPMG [SEQ ID NO: 22]and in which:v) CDR 2 is an amino acid sequence that is chosen from the groupconsisting of the following amino acid sequences:

SISWSGTYTAYSDNVKG [SEQ ID NO: 23] GISWSGVSTDYAEFAKG [SEQ ID NO: 24]TSISWSGSYTAYADNVKG [SEQ ID NO: 25] SISWSGMSTYYTDSVKG [SEQ ID NO: 26]TITSGGRTSYADSVKG [SEQ ID NO: 27] AISWSGGLTYYADSVKG [SEQ ID NO: 28]TITSGGSTNYADPVKG [SEQ ID NO: 29] TITSGGSTNYADSVKG [SEQ ID NO: 30]AISRTGGSTYYARSVEG [SEQ ID NO: 31] AISRTGGSTYYPDSVEG [SEQ ID NO: 32]and in which:vi) CDR 3 is an amino acid sequence that is chosen from the groupconsisting of the following amino acid sequences:

QSRYRSNYYDHDDKYAY [SEQ ID NO: 33] LGRYRSNWRNIGQYDY [SEQ ID NO: 34]QSRYSSNYYDHDDKYAY [SEQ ID NO: 35] SNRYRTHTTQAMYNY [SEQ ID NO: 36]VVDGKRAP [SEQ ID NO: 37] NRRQKTVQMGERAYDY [SEQ ID NO: 38] NLKQGSYGYRFNDY[SEQ ID NO: 39] NLKQGDYGYRFNDY [SEQ ID NO: 40] AGVRAEDGRVRTLPSEYNF [SEQID NO: 41] AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42] AGVRAEDGRVRSLPSEYTF [SEQID NO: 43]

Preferably, in the Nanobodies of the invention according to the latteraspect:

When CDR1 is: NYGMG [SEQ ID NO: 15]; then CDR2 is: SISWSGTYTAYSDNVKG[SEQ ID NO: 23]; and CDR3 is: QSRYRSNYYDHDDKYAY [SEQ ID NO: 33] WhenCDR1 is: SYTLG [SEQ ID NO: 16]; then CDR2 is: GISWSGVSTDYAEFAKG [SEQ IDNO: 24]; and CDR3 is: LGRYRSNWRNIGQYDY [SEQ ID NO: 34] When CDR1 is:NYGMG [SEQ ID NO: 15]; then CDR2 is: TSISWSGSYTAYADNVKG [SEQ ID NO: 25];and CDR3 is: QSRYSSNYYDHDDKYAY [SEQ ID NO: 35] When CDR1 is: NYNMG [SEQID NO: 17]; then CDR2 is: SISWSGMSTYYTDSVKG [SEQ ID NO: 26]; and CDR3is: SNRYRTHTTQAMYNY [SEQ ID NO: 36] When CDR1 is: SSAMA [SEQ ID NO: 18];then CDR2 is: TITSGGRTSYADSVKG [SEQ ID NO: 27]; and CDR3 is: VVDGKRAP[SEQ ID NO: 37] When CDR1 is: YYNTG [SEQ ID NO: 19]; then CDR2 is:AISWSGGLTYYADSVKG [SEQ ID NO: 28]; and CDR3 is: NRRQKTVQMGERAYDY [SEQ IDNO: 38] When CDR1 is: IGAMG [SEQ ID NO: 20]; then CDR2 is:TITSGGSTNYADPVKG [SEQ ID NO: 29]; and CDR3 is: NLKQGSYGYRFNDY [SEQ IDNO: 39] When CDR1 is: IGAMG [SEQ ID NO: 20]; then CDR2 is:TITSGGSTNYADSVKG [SEQ ID NO: 30]; and CDR3 is: NLKQGSYGYRFNDY [SEQ IDNO: 39] When CDR1 is: IGAMG [SEQ ID NO: 20]; then CDR2 is:TITSGGSTNYADSVKG [SEQ ID NO: 30]; and CDR3 is: NLKQGDYGYRFNDY [SEQ IDNO: 40] When CDR1 is: IGTMG [SEQ ID NO: 21]; then CDR2 is:TITSGGSTNYADSVKG [SEQ ID NO: 30]; and CDR3 is: NLKQGDYGYRFNDY [SEQ IDNO: 40] When CDR1 is: YNPMG [SEQ ID NO: 22]; then CDR2 is:AISRTGGSTYYARSVEG [SEQ ID NO: 31]; and CDR3 is: AGVRAEDGRVRTLPSEYNF [SEQID NO: 41] When CDR1 is: YNPMG [SEQ ID NO: 22]; CDR2: AISRTGGSTYYPDSVEG[SEQ ID NO: 32]; and CDR3 is: AGVRAEDGRVRTLPSEYTF [SEQ ID NO: 42] WhenCDR1 is: YNPMG [SEQ ID NO: 22]; then CDR2 is: AISRTGGSTYYPDSVEG [SEQ IDNO: 32]; and CDR3 is: AGVRAEDGRVRSLPSEYTF [SEQ ID NO: 43]

In particular, according to one preferred, but non-limiting aspect ofthe aspect of the invention, a Nanobody can generally be defined as apolypeptide comprising an amino acid sequence that is comprised of fourframework regions/sequences interrupted by three complementaritydetermining regions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, G, Q, R,    S, L; and is preferably chosen from the group consisting of G, E or    Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, G, Q, R,    S, L; and is preferably chosen from the group consisting of G, E or    Q;    and in which:

-   ii) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:

-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which

-   iv) the amino acid residue at position 108 according to the Kabat    numbering is Q; and in which:

-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:

-   ii) the amino acid residue at position 45 according to the Kabat    numbering is R; and in which:

-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:

-   iv) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:

-   ii) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:

-   iii) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:

-   iv) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:

-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to a-1) to a-4)above; according to b) above; according to b-1) to b-4) above; accordingto c) above; and/or according to c-1) to c-4) above, in which;

-   a) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    or in which:-   b) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q;    and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, I, V or F, and is most preferably F.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified is on the basis of thefollowing three groups:

-   a) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW at    positions 44-47 according to the Kabat numbering and Q at position    108 according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a V at position 37, and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. The GLEW group also comprises some GLEW-like sequences    such as those mentioned in Table 2 below;-   b) The “KERE-group”: Nanobodies with the amino acid sequence KERE or    KQRE or at positions 43-46 according to the Kabat numbering and Q or    L at position 108 according to the Kabat numbering. As further    described herein, Nanobodies within this group usually have a F at    position 37, an L or F at position 47; and can have a W, P, R or S    at position 103, and preferably have a W at position 103;-   c) The “103 P, R, S-group”: Nanobodies with a P R or S at    position 103. These Nanobodies can have either the amino acid    sequence GLEW at positions 44-47 of the Kabat numbering or the amino    acid sequence KERE or KQRE at positions 43-46 according to the Kabat    numbering, the latter most preferably in combination with an F at    position 37 and an L or an F at position 47 (as defined for the    KERE-group); and can have Q or L at position 108 according to the    Kabat numbering, and preferably have Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred definitionsabove, and are more preferably as defined according to one of the morepreferred definitions above.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred definitionsabove, and are more preferably as defined according to one of the morepreferred definitions above.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferreddefinitions above, and are more preferably as defined according to oneof the more preferred definitions above.

Also, more generally and in addition to the 108Q, 43E/44R and 103P,R,Sresidues mentioned above, the Nanobodies of the invention can contain,at one or more positions that, in a conventional V_(H) domain, wouldform (part of) the V_(H)/V_(L) interface, contain one or more amino acidresidues that are more highly charged than the amino acid residues thatnaturally occur at the same position(s) in the corresponding naturallyoccurring V_(H) or V_(HH) domain, and in particular one or more chargedamino acid residues (as mentioned in Table 1).

Such substitutions include, but are not limited to the GLEW-likesequences mentioned in Table 2 below; as well as the substitutions thatare described in the International Application WO 00/29004 for so-called“microbodies”, e.g. a Q at position 108 and KLEW at positions 44-47.

In the Nanobodies of the invention, the amino acid residue at position83 is chosen from the group consisting of L, M, S, V and W; and ispreferably L.

Also, in the Nanobodies of the invention, the amino acid residue atposition 83 is chosen from the group consisting of R, K, N, E, I and Q;and is most preferably either K or E (for Nanobodies corresponding tonaturally occurring V_(HH) domains) or R (for “humanized” Nanobodies, asdescribed below). The amino acid residue at position 84 is chosen fromthe group consisting of P, A, R, S, D and V, and is most preferably P(for Nanobodies corresponding to naturally occurring V_(HH) domains) orR (for “humanized” Nanobodies, as described below).

Furthermore, in the Nanobodies of the invention, the amino acid residueat position 104 is chosen from the group consisting of G and D; and ismost preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human VH domain, VH3, aresummarized in Table 2.

Some especially preferred combinations of these Hallmark Residues asoccur in naturally occurring V_(HH) domains are mentioned in Table 3.For comparison, the corresponding amino acid residues of the humanV_(H)3 called DP-47 have been indicated in italics.

TABLE 2 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; L, M, S, V, W; preferably L predominantly L  37 V, I,F; usually V F⁽¹⁾, Y, H, I or V, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G G⁽²⁾,E⁽³⁾, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾ orE⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S or Y; preferably W⁽²⁾,L⁽¹⁾, F⁽¹⁾ or R  83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, I, M or Q;preferably K or R; most preferably K  84 A, T, D; P⁽⁵⁾, A, L, R, S, D,V; preferably P predominantly A 103 W W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W104 G G or D; preferably G 108 L, M or T; Q, L⁽⁷⁾ or R; preferably Q orL⁽⁷⁾ predominantly L Notes: ⁽¹⁾In particular, but not exclusively, incombination with KERE or KQRE at positions 43-46. ⁽²⁾Usually as GLEW atpositions 44-47. ⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. asKEREL, KEREF, KQREL, KQREF or KEREG at positions 43-47. Alternatively,also sequences such as TERE (for example TEREL), KECE (for example KECELor KECER), RERE (for example REREG), QERE (for example QEREG), KGRE (forexample KGREG), KDRE (for example KDREV) are possible. Some otherpossible, but less preferred sequences include for example DECKL andNVCEL. ⁽⁴⁾With both GLEW at positions 44-47 and KERE or KQRE atpositions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturallyoccurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, incombination with GLEW at positions 44-47. ⁽⁷⁾With the proviso that whenpositions 44-47 are GLEW, position 108 is always Q. ⁽⁸⁾The GLEW groupalso contains GLEW-like sequences at positions 44-47, such as forexample GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLERand ELEW.

TABLE 3 Some preferred combinations of Hallmark Residues in naturallyoccurring Nanobodies. For humanization of these combinations, referenceis made to the specification. 11 37 44 45 47 83 84 103 104 108 DP-47(human) M V G L W R A W G L “KERE” group L F E R L K P W G Q L F E R F EP W G Q L F E R F K P W G Q L Y Q R L K P W G Q L F L R V K P Q G Q L FQ R L K P W G Q L F E R F K P W G Q “GLEW” group L V G L W K S W G Q M VG L W K P R G Q

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables 4-7mention some non-limiting residues that can be present at each position(according to the Kabat numbering) of the FR1, FR2, FR3 and FR4 ofnaturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering Chothia (supra) that the residues at thesepositions already form part of CDR1.)

In Tables 4-7, some of the non-limiting residues that can be present ateach position of a human V_(H)3 domain have also been mentioned. Again,for each position, the amino acid residue that most frequently occurs ateach position of a naturally occurring human V_(H)3 domain is indicatedin bold; and other preferred amino acid residues have been underlined.

TABLE 4 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table 2) Amino acid residue(s): Pos.Human V_(H)3 Camelid V_(HH)'s  1 E, Q Q, A, E, D, H, R  2 V V, A, E, G,L, M, Q  3 Q Q, K, E, H, P, R, Y  4 L L, F, P, R, V  5 V, L Q, E, L, V,M, P, A, I  6 E E, D, Q, A, H  7 S, T S, F, H  8 G, R G, A, R  9 G G, E10 G, V G, D, R, A, E, N, T, V 11 Hallmark residue: L, M, S, V, W, F, N,P, T, Y; preferably L 12 V, I V, A, G, M 13 Q, K, R Q, E, K, D, G, A, H,L, N, P. R, T 14 P A, Q, A, G, P, T, V, E, F, I, N, S 15 G G 16 G, R G,A, E, D, N, P, R, S, V, W 17 S S, F, T, N, P, A, C 18 L L, V, M, Q, R 19R, K R, K, L, N, S, T, A, F, G, I, M, Q 20 L L, F, I, V, M, S 21 S S, F,T, G, H, P, A 22 C C 23 A, T A, D, P, S, T, V, E, G, I, L, Q, R 24 A A,I, S, T, V, C, E, F, G, L, N, P, Q, Y 25 S S, A, F, P, T, L, V 26 G G,D, E, R, S, V, A, I, M, P, T 27 F S, F, R, L, P, G, N, A, D, E, H, I, K,M, Q, T, V, Y 28 T N, T, E, D, S, I, R, A, G, R, F, Y, L, M, P, V 29 F,V F, L, D, S, I, G, V, A, E, P, T, Y 30 S, D, G N, S, E, G, A, D, M, T,H, I, P, R, V, W

TABLE 5 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table 2) Amino acid residue(s): Pos.Human V_(H)3 Camelid V_(HH)'s 36 W W 37 Hallmark residue: F⁽¹⁾, Y, H, I,A, L, P, S or V preferably F⁽¹⁾ or Y 38 R R 39 Q Q, H, P, R, A, D, G, L,E 40 A A, F, G, P, T, V, I, L, N, R, S, Y 41 P, S, T P, A, L, S, I, Q, T42 G G, E, D, R, T, V 43 K K, D, E, N, Q, R, T, V, A, L, M, S 44Hallmark residue: G⁽²⁾, E⁽³), D, Q, R, S, L, A, F, K, M, N, P, V, W, Y;preferably G⁽²⁾, E⁽³⁾, or Q; most preferably G⁽²⁾ or E⁽³⁾ 45 Hallmarkresidue: L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V, D, E, G, H, K, T; preferably L⁽²⁾or R⁽³⁾ 46 E, V E, D, K, Q, V, A, G, N 47 Hallmark residue: W⁽²⁾, L⁽¹⁾or F⁽¹⁾, A, G, I, M, R, S, D, E, H, K, Q, T, V or Y; preferably W⁽²⁾,L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L, A, C, E, F, G, H, M, P, Q, R, S, T, V, W,Y 49 S, A, G A, S, A, G, T, V, D, E, I, L, Q, R, Y

TABLE 6 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table 2) Amino acid residue(s): Pos.Human V_(H)3 Camelid V_(HH)'s 66 R R 67 F F, L, V, A, D, I, S, Y 68 T T,A, S, D, F, G, I, K, N 69 I I, M, V, A, F, L, R, S, T 70 S S, A, F, E,G, K, P, T, V 71 R R, G, I, K, Q, S, T, W, A, F, L, M, N 72 D, E D, E,G, N, V, A, H, I, L, Q, S, T 73 N, D, G N, D, F, I, K, S, T, Y, A, G, H,L, M, R, V 74 A, S A, D, G, N, P, S, T, F, H, I, L, R, V, Y 75 K K, A,E, K, L, N, Q, R, D, G, I, M, S, T, V, W 76 N, S N, D, K, R, S, T, Y, E,G, H, I, Q 77 S, T, I T, A, E, I, M, S, K, L, N, R, V 78 L, A V, L, A,F, G, I, M, E, N, Q, R, S, T, W 79 Y, H Y, A, D, F, H, S, T, C, E, I, L,N, V, W 80 L L, F, V, M 81 Q Q, E, R, T, G, H, I, K, L, M, N 82 M M, I,L, V, G, P, T  82a N, G N, D, G, H, S, T, A, E, I, K, R, V  82b S S, N,D, G, R, A, C, E, F, I, K, M, P, T, V  82c L L, P, M, T, V 83 Hallmarkresidue: R, K⁽⁵⁾, N, E⁽⁵⁾, I, M, A, D, G, L, Q, S, T or Q; preferably Kor R; most preferably K 84 Hallmark residue: P⁽⁵⁾, A, L, R, S, D, V, F,G, H, N, T, Y; preferably P 85 E, G E, D, G, Q, A, N, R, V, Y 86 D D, E,F, Y 87 T, M T, S, A, C, M 88 A A, G, S, D, L, N, P 89 V, L V, A, D, I,L, M, N, R, T, E, F, S 90 Y Y, F, E, H, N 91 Y, H Y, D, F, H, L, S, T,V, C, I, N, R, W 92 C C 93 A, K, T A, N, G, H, K, R, S, T, V, Y, E, F,I, L, M, Q 94 K, R, T A, V, C, F, G, I, L, R, S, D, E, K, M, N, P, Q, T,W, Y T or K;

TABLE 7 Non-limiting examples of amino acid residues in FR4 Amino acidresidue(s): Pos. Human V_(H)3 Camelid V_(HH)'s 103 Hallmark residue:W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S, F, G, K, L, N, Q, V, Y; preferably W 104 Hallmarkresidue: G, A, R, S, T or D; preferably G 105 Q, R Q, E, K, P, R, G, H,L, S, V 106 G G 107 T T, A, I, N, P 108 Hallmark residue: Q, L⁽⁷⁾, E, H,N, P, T or R; preferably Q or L⁽⁷⁾ 109 V V 110 T T, I, A 111 V V, A, L,G 112 S S, F, A, L, P, T, Y 113 S S, A, L, P, F, T (for the footnotes,see the footnotes to Table 2)

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:

-   i) the Hallmark residues are as defined herein; and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:        and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[1] QVQLQESGGG X VQAGGSLRLSCAASG [26] [SEQ ID NO: 1]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   ii) FR2 is chosen from the group consisting of the amino acid    sequence:

[36] W X RQAPGK XX E X VA [49] [SEQ ID NO: 2]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 3] [66] RFTISRDNAKNTVYLQMNSL XX EDTAVYYCAA [94]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequence:

[103]  XX QGT X VTVSS [113] [SEQ ID NO: 4]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and in which:

-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above;    in which the Hallmark Residues are indicated by “X” and are as    defined hereinabove and in which the numbers between brackets refer    to the amino acid positions according to the Kabat numbering.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which: and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[1] QVQLQESGGG L VQAGGSLRLSCAASG [26] [SEQ ID NO: 5]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residue at position is as indicated in the            sequence above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residue at position is as indicated in the            sequence above; and in which:

-   ii) FR2 is chosen from the group consisting of the amino acid    sequences:

[36] W F RQAPGK ER E L VA [49] [SEQ ID NO: 6] [36] W F RQAPGK ER E F VA[49] [SEQ ID NO: 7] [36] W F RQAPGK ER E G A [49] [SEQ ID NO: 8] [36] WF RQAPGK QR E L VA [49] [SEQ ID NO: 9] [36] W F RQAPGK QR E F VA [49][SEQ ID NO: 10] [36] W Y RQAPGK GL E W A [49] [SEQ ID NO: 11]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are            as indicated in each of the sequences above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are            as indicated in each of the sequences above;            and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSL KP EDTAVYYCAA [94]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with the above amino acid sequence; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 83 and 84 are as            indicated in each of the sequences above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 83 and 84 are as            indicated in each of the sequences above;            and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequences:

[103]  WG QGT Q VTVSS [113] [SEQ ID NO: 13] [103]  WG QGT L VTVSS [113][SEQ ID NO: 14]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequence; in        which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 103, 104 and 108 are            as indicated in each of the sequences above;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 103, 104 and 108 are            as indicated in each of the sequences above;            and in which:

-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:        and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[1] QVQLQESGGG L VQAGGSLRLSCAASG [26] [SEQ ID NO: 5]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residue at position is as indicated in the            sequence above;            and in which:

-   ii) FR2 is chosen from the group consisting of the amino acid    sequences:

[36] W F RQAPGK ER E L VA [49] [SEQ ID NO: 6] [36] W F RQAPGK ER E F VA[49] [SEQ ID NO: 7] [36] W F RQAPGK ER E G A [49] [SEQ ID NO: 8] [36] WF RQAPGK QR E L VA [49] [SEQ ID NO: 9] [36] W F RQAPGK QR E F VA [49][SEQ ID NO: 10] [36] W Y RQAPGK GL E W A [49] [SEQ ID NO: 11]

-   -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are            as indicated in each of the sequences above;            and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66] RFTISRDNAKNTVYLQMNSL KP EDTAVYYCAA [94]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 83 and 84 are as            indicated in each of the sequences above;            and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequences:

[103]  WG QGT Q VTVSS [113] [SEQ ID NO: 13] [103]  WG QGT L VTVSS [113][SEQ ID NO: 14]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 7; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 103, 104 and 108 are            as indicated in each of the sequences above;            and in which:

-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:        and in which

-   i) FR1 is chosen from the group consisting of the amino acid    sequence:

[1] QVQLQESGGG L VQAGGSLRLSCAASG[26] [SEQ ID NO: 5]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residue at position is as indicated in the            sequence above;            and in which:

-   ii) FR2 is chosen from the group consisting of the amino acid    sequence:

[36] W Y RQAPGK GL E W A [49] [SEQ ID NO: 11]

-   -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are            as indicated in each of the sequences above;            and in which:

-   iii) FR3 is chosen from the group consisting of the amino acid    sequence:

[SEQ ID NO: 12] [66]RFTISRDNAKNTVYLQMNSL KP EDTAVYYCAA[94]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 83 and 84 are as            indicated in each of the sequences above;            and in which:

-   iv) FR4 is chosen from the group consisting of the amino acid    sequence:

[103] WG QGT Q VTVSS[113] [SEQ ID NO: 13]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 7; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequence(s);            and        -   (3) the Hallmark residues at positions 103, 104 and 108 are            as indicated in each of the sequences above;            and in which:

-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

In another preferred, but not limiting aspect, a Nanobody of theinvention can have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which:        and in which

-   i) FR1 is chosen from the group consisting of the FR1 sequences    present in the Nanobodies of SEQ ID NO's 60 to 73 and SEQ ID NO's 86    to 97, and in particular in the humanized Nanobodies of SEQ ID NO's    86 to 97,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR1 sequences; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR1 sequence; and        -   (3) the Hallmark residue at position is as indicated in said            FR1 sequence;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of said FR1 sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 4; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR1 sequence; and        -   (3) the Hallmark residue at position is as indicated in said            FR1 sequence;            and in which:

-   ii) FR2 is chosen from the group consisting of the FR2 sequences    present in the Nanobodies of SEQ ID NO's 60 to 73 and SEQ ID NO's 86    to 97, and in particular in the humanized Nanobodies of SEQ ID NO's    86 to 97,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR2 sequences; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR2 sequence; and        -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are            as indicated in said FR2 sequence;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of said FR2 sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 5; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR2 sequence; and        -   (3) the Hallmark residues at positions 37, 44, 45 and 47 are            as indicated in said FR2 sequence;            and in which:

-   iii) FR3 is chosen from the group consisting of the FR3 sequences    present in the Nanobodies of SEQ ID NO's 60 to 73 and SEQ ID NO's 86    to 97, and in particular in the humanized Nanobodies of SEQ ID NO's    86-97,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR3 sequences; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR3 sequence; and        -   (3) the Hallmark residues at positions 83 and 84 are as            indicated in said FR3 sequence;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of said FR3 sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR3 sequence; and        -   (3) the Hallmark residues at positions 83 and 84 are as            indicated in said FR3 sequence;            and in which:

-   iv) FR4 is chosen from the group consisting of the FR4 sequences    present in the Nanobodies of SEQ ID NO's 60 to 73 and SEQ ID NO's 86    to 97, and in particular in the humanized Nanobodies of SEQ ID NO's    86 to 97,    -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of said FR4 sequences; in which        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR4 sequence; and        -   (3) the Hallmark residues at positions 103, 104 and 108 are            as indicated in said FR3 sequence;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of said FR4 sequences, in which:        -   (1) any amino acid substitution at any position other than a            Hallmark position is preferably either a conservative amino            acid substitution (as defined herein) and/or an amino acid            substitution as defined in Table 6; and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to said FR4 sequence; and        -   (3) the Hallmark residues at positions 103, 104 and 108 are            as indicated in said FR4 sequence;            and in which:

-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred definitions above, and are    more preferably as defined according to one of the more preferred    definitions above.

Some particularly preferred Nanobodies of the invention can be chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's 60to 73 and SEQ ID NO's 86 to 97, and in particular in the humanizedNanobodies of SEQ ID NO's 86 to 97 or from the group consisting of aminoacid sequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity (as defined herein) with one of the amino acid sequences of SEQID NO's 60 to 73 and SEQ ID NO's 86 to 97 (and preferably of SEQ ID NO's86 to 97); in which

-   -   (1) the Hallmark residues can be as indicated in Table 2 above;    -   (2) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Tables 4-7; and/or    -   (3) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequence(s).

Some even more particularly preferred Nanobodies of the invention can bechosen from the group consisting of the amino acid sequences of SEQ IDNO's 60 to 73 and SEQ ID NO's 86 to 97, and in particular in thehumanized Nanobodies of SEQ ID NO's 86 to 97 or from the groupconsisting of amino acid sequences that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity (as defined herein) with one of the amino acidsequences of SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to 97 (andpreferably of SEQ ID NO's 86 to 97); in which

-   -   (1) the Hallmark residues are as indicated in the pertinent        sequence chosen from SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to        97 (and preferably from SEQ ID NO's 86 to 97);    -   (2) any amino acid substitution at any position other than a        Hallmark position is preferably either a conservative amino acid        substitution (as defined herein) and/or an amino acid        substitution as defined in Tables 4-7; and/or    -   (3) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the pertinent sequence chosen from SEQ ID NO's 60 to        73 and SEQ ID NO's 86 to 97 (and preferably from SEQ ID NO's 86        to 97).

Some of the most preferred Nanobodies of the invention can be chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's 60to 73 and SEQ ID NO's 86 to 97, and in particular from the humanizedNanobodies of SEQ ID NO's 86 to 97.

As will be clear from the above, the term Nanobodies of the invention asused herein in its broadest sense also comprises natural or syntheticmutants, variants, alleles, analogs and orthologs (hereinbelowcollectively referred to as “analogs”) of the Nanobodies mentioned inthe SEQ ID NO's 60 to 73 and SEQ ID NO's 86 to 97.

Generally, such analogs can for example comprise homologous sequences,functional portions, or a functional portion of a homologous sequence(as further defined below) of a Nanobody. Generally, in such analogs,each amino acid residue (other than the Hallmark Residue) in each of theframework regions can be replaced by any other amino acid residue,provided that the total degree of sequence identity of the frameworkregions remains as defined herein. Preferably, however, in such analogs:

-   -   one or amino acid residues in the above framework sequences are        replaced by one or more amino acid residues that naturally occur        at the same position in a naturally occurring V_(HH) domain.        Some examples of such substitutions are mentioned in Tables 4-7        above;        and/or:    -   one or amino acid residues in the above framework sequences are        replaced by one or more amino acid residues that can be        considered a “conservative” amino acid substitution, as        described hereinabove;        and/or:    -   one or amino acid residues in the above framework sequences are        replaced by one or more amino acid residues that naturally occur        at the same position in a naturally occurring V_(H) domain of a        human being. This is generally referred to as “humanization” of        the naturally occurring V_(HH)/Nanobody in general and of said        position in particular, and will be discussed in more detail        hereinbelow;        and:    -   positions for which only one amino acid residue is mentioned for        both the V_(H) domain and the V_(HH) domain in Tables 4-7 above        are preferably not replaced.

Also, although generally less preferred, in such analogs, one or moreamino acid residues may be deleted from the framework regions and/orinserted into the framework regions (optionally in addition to one ormore amino acid substitutions as mentioned above), provided that thetotal degree of sequence identity of the framework regions remains asdefined herein. The Hallmark residues should not be deleted. Also, mostpreferably, amino acid residues for which only one amino acid residue ismentioned for both the V_(H) domain and the V_(HH) domain in Tables 4-7above are preferably not deleted.

Preferably, such analogs should be such that they still can bind to,have affinity for and/or have specificity for vWF, i.e. with an affinityand/or a specificity which is at least 10%, preferably at least 50%,more preferably at least 70%, even more preferably at least 80%, such asat least 90%, at least 95%, at least 99% or more, of the affinity and/orspecificity of at least one of the Nanobodies of SEQ ID No's 60 to 73and SEQ ID NO's 86 to 97, as determined using a suitable assay, forexample an assay to determine binding of the analog to vWF, and inparticular one of the assays as used in the Examples below.

Generally, such analogs can for example be obtained by providing anucleic acid that encodes a naturally occurring V_(HH) domain, changingthe codons for the one or more amino acid residues that are to behumanized into the codons for the corresponding human amino acidresidue(s), expressing the nucleic acid/nucleotide sequence thusobtained in a suitable host or expression system; and optionallyisolating and/or purifying the analog thus obtained to provide saidanalog in essentially isolated form (as defined hereinabove). This cangenerally be performed using methods and techniques known per se, whichwill be clear to the skilled person, for example from the handbooks andreferences cited herein and/or from the further description hereinbelow.Alternatively, and for example, a nucleic acid encoding an analog can besynthesized in a manner known per se (for example using an automatedapparatus for synthesizing nucleic acid sequences with a predefinedamino acid sequence) and can be expressed in a suitable host orexpression system, upon which the analog thus obtained can optionally beisolated and/or purified so as to provide said analog in essentiallyisolated form (as defined hereinabove). Another way to provide theanalogs involves chemical synthesis of the pertinent amino acid sequenceusing techniques for peptide synthesis known per se, such as thosementioned hereinbelow.

It will be also generally be clear to the skilled person that Nanobodies(including analogs thereof) can also be prepared starting from humanV_(H) sequences (i.e. amino acid sequences or the correspondingnucleotide sequences), such as for example human V_(H)3 sequences suchas DP-47, DP-51 or DP-29, by changing one or more amino acid residues inthe amino acid sequence of said human V_(H) domain, so as to provide anamino acid sequence that has (a) a Q at position 108; and/or (b) E atposition 44 and/or R at position 45, and preferably E at position 44 andR at position 45; and/or (c) P, R or S at position 103, as describedabove. Again, this can generally be performed using the various methodsand techniques referred to in the previous paragraph, using an aminoacid sequence and/or nucleotide sequence for a human V_(H) domain as astarting point.

The term Nanobodies as used herein in its broadest sense also comprisesparts or fragments of the Nanobodies (including analogs) of theinvention as defined herein, which can again be as further describedbelow.

Generally, parts or fragments of the Nanobodies and/or analogs haveamino acid sequences in which, compared to the amino acid sequence ofthe corresponding full length Nanobody or analog, one or more of theamino acid residues at the N-terminal end, one or more amino acidresidues at the C-terminal end, one or more contiguous internal aminoacid residues, or any combination thereof, have been deleted and/orremoved. It is also possible to combine one or more of such parts orfragments to provide a Nanobody of the invention.

Preferably, the amino acid sequence of a Nanobody that comprises one ormore parts or fragments of a full length Nanobody and/or analog shouldhave a degree of sequence identity of at least 50%, preferably at least60%, more preferably at least 70%, such as at least 80%, at least 90% orat least 95%, with the amino acid sequence of the corresponding fulllength Nanobody.

Also, the amino acid sequence of a Nanobody that comprises one or moreparts or fragments of a full length Nanobody and/or analog is preferablysuch that is comprises at least 10 contiguous amino acid residues,preferably at least 20 contiguous amino acid residues, more preferablyat least 30 contiguous amino acid residues, such as at least 40contiguous amino acid residues, of the amino acid sequence of thecorresponding full length Nanobody.

Generally, such parts or fragments of the Nanobodies of the inventionwill have amino acid sequences in which, compared to the amino acidsequence of the corresponding full length Nanobody of the invention, oneor more of the amino acid residues at the N-terminal end, one or moreamino acid residues at the C-terminal end, one or more contiguousinternal amino acid residues, or any combination thereof, have beendeleted and/or removed. It is also possible to combine one or more ofsuch parts or fragments to provide a Nanobody of the invention.

According to one preferred embodiment, a fragment as used hereincomprises at least one of the CDR's present in a full-sized Nanobody ofthe invention, preferably at least two of the CDR's present in afull-sized Nanobody of the invention, more preferably at least CDR2 andCDR3 present in a full-sized Nanobody of the invention, such as forexample all three CDR's present in a full-sized Nanobody of theinvention.

According to another particularly preferred, but non-limitingembodiment, such a part or fragment comprises at least FR3, CDR3 and FR4of the corresponding full length Nanobody of the invention, i.e. as forexample described in the International application WO 03/050531 (Lasterset al.).

Preferably, such parts or fragments should be such that they still canbind to, have affinity for and/or have specificity for vWF, i.e. with anaffinity and/or a specificity which is at least 10%, preferably at least50%, more preferably at least 70%, even more preferably at least 80%,such as at least 90%, at least 95%, at least 99% or more, of theaffinity and/or specificity of the corresponding full-sized Nanobody ofthe invention, for example an assay to determine binding of the analogto vWF, and in particular one of the assays as used in the Examplesbelow.

From the description hereinabove, it will be clear that the amino acidsequences of the Nanobodies used herein differ at least one amino acidposition in at least one of the framework regions from the amino acidsequences of naturally occurring V_(H) domains, such as the amino acidsequences of naturally occurring V_(H) domains of antibodies from humanbeings. In particular, it will be clear that the amino acid sequences ofthe Nanobodies used herein differ at least one of the Hallmark Residuesfrom amino acid sequences of naturally occurring V_(H) domains, such asthe amino acid sequences of naturally occurring V_(H) domains fromantibodies from Camelids and/or human beings.

Thus, according to one specific embodiment, a Nanobody of the inventionhas an amino acid sequence that differs at least one amino acid positionin one of the framework regions from the amino acid sequence of anaturally occurring V_(H) domain. According to a more specific, butnon-limiting embodiment of the invention, a Nanobody of the inventionhas an amino acid sequence that differs at least one of the Hallmarkresidues from the amino acid sequence of a naturally occurring V_(H)domain.

From the description hereinabove, it will also be clear that the aminoacid sequences of the some of the Nanobodies of the invention, such asthe humanized Nanobodies of the invention, will differ at least oneamino acid position in at least one of the framework regions (i.e.either at the position of a Hallmark residue or at another position)from the amino acid sequences of naturally occurring V_(HH) domains.Thus, according to one specific, but non-limiting embodiment, a Nanobodyof the invention has an amino acid sequence that differs at at least oneamino acid position in one of the framework regions from the amino acidsequence of a naturally occurring V_(HH) domain. According to a morespecific, but non-limiting embodiment of the invention, a Nanobody ofthe invention has an amino acid sequence that differs at least one ofthe Hallmark residues from the amino acid sequence of a naturallyoccurring V_(HH) domain.

As mentioned above, the invention also relates to proteins orpolypeptides comprising at least one V_(HH) domain (i.e. as identifiedusing the methods of the invention) or at least one Nanobody basedthereon.

According to one non-limiting embodiment of the invention, such apolypeptide of the invention essentially consists of a Nanobody. By“essentially consist of” is meant that the amino acid sequence of thepolypeptide of the invention either is exactly the same as the aminoacid sequence of a Nanobody (as mentioned above) or corresponds to theamino acid sequence of a Nanobody in which a limited number of aminoacid residues, such as 1-10 amino acid residues and preferably 1-6 aminoacid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, have beenadded to the amino terminal end, to the carboxy terminal end, or both tothe amino terminal end and to the carboxy terminal end of the amino acidsequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, said amino acidresidues may:

-   a) form a “tag”, i.e. an amino acid sequence or residue that allows    or facilitates the purification of the Nanobody, for example using    affinity techniques directed against said sequence or residue.    Thereafter, said sequence or residue may be removed (e.g. by    chemical or enzymatical cleavage) to provide the nucleotide sequence    of the invention (for this purpose, the sequence or residue may    optionally be linked to the amino acid sequence of the invention via    a cleavable linker sequence). Some preferred, but non-limiting    examples of such residues are multiple histidine residues and    glutatione residues,-   b) can be a N-terminal Met residue, for example as result of    expression in a heterologous host cell or host organism.-   c) may be one or more amino acid residues that can be provided with    functional groups and/or that have been functionalized, in a manner    known per se. For example, as is known in the art, amino acid    residues such as lysine and in particular cysteine allow for the    attachment of PEG groups, which may mask surface site on a protein    and thus for example decrease immunogenicity, improve half-life in    plasma and stabilize against proteolytic cleavage;-   d) increase the half-life in serum of a Nanobody or polypeptide of    the invention. Amino acid sequences that can be attached to and/or    fused with therapeutic proteins in order to increase their half-life    in vivo are well know to the skilled person and include human serum    proteins or fragments thereof (such as human serum albumin or a part    or fragment thereof), or even Fc portions of antibodies (in    particular of human antibodies). Also, as already described herein,    such an amino acid sequence for increasing the half-life may be an    amino acid sequence directed against a serum protein, such as a    Nanobody directed against a serum protein, for example against human    serum albumin.

With regard to pegylation, its should be noted that generally, theinvention also encompasses any Nanobody of the invention and/orpolypeptide of the invention that has been pegylated at one or moreamino acid positions, preferably in such a way that said pegylationeither (1) increases the half-life in vivo; (2) reduces immunogenicity;(3) provides one or more further beneficial properties known per se forpegylation; (4) does not essentially affect the affinity of the Nanobodyand/or polypeptide for vWF (e.g. does not reduce said affinity by morethan 90%, preferably not by more than 50%, and more preferably not bymore than 10%, as determined by a suitable assay, such as thosedescribed in the Examples below); and/or (4) does not affect any of theother desired properties of the Nanobodies and/or polypeptides of theinvention. Suitable PEG-groups and methods for attaching them, eitherspecifically or non-specifically, will be clear to the skilled person.Suitable kits and reagents for such pegylation can for example beobtained from Nektar (CA, USA).

According to one non-limiting embodiment, one or more amino acidresidues can be added to, inserted in and/or substituted in the aminoacid sequence of a Nanobody or polypeptide of the invention, so as toprovide one or more specific amino acid residues for attachment of aPEG-group.

The invention also encompasses any Nanobody of the invention and/orpolypeptide of the invention that has been glycosylated at one or moreamino acid positions, usually depending upon the host used to expressthe Nanobody or polypeptide of the invention (as further describedbelow).

According to one non-limiting embodiment, one or more amino acidresidues can be added to, inserted in and/or substituted in the aminoacid sequence of a Nanobody or polypeptide of the invention, so as toprovide one or more specific amino acid residues and/or a site that canbe glycosylated by the host organism used. By means of a preferred, butnon-limiting example, the N-residue on position 50 within CDR2 of aNanobody of the invention can for example be replaced by a Q, D or Sresidue so as to provide a glycosylation site, e.g. for glycosylation byPichia.

According to another embodiment, a polypeptide of the invention cancomprise a the amino acid sequence of a Nanobody, which is fused at itsamino terminal end, at its carboxy terminal end, or both at its aminoterminal end and at its carboxy terminal end with at least one furtheramino acid sequence.

Again, said further amino acid sequence(s) may or may not change, alteror otherwise influence the (biological) properties of the Nanobody andmay or may not add further functionality to the Nanobody.

For example, according to one preferred, but non-limiting embodiment,said further amino acid sequence may comprise at least one furtherNanobody, so as to provide a polypeptide of the invention that comprisesat least two, such as three, four or five, Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein).

Polypeptides of the invention comprising two or more Nanobodies willalso referred to herein as “multivalent” polypeptides. For example a“bivalent” polypeptide of the Invention comprises two Nanobodies,optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three Nanobodies, optionallylinked via two linker sequences; etc.

In a multivalent polypeptide of the invention, the two or moreNanobodies may be the same or different. For example, the two or moreNanobodies in a multivalent polypeptide of the invention:

-   -   may be directed against the same antigen, i.e. against the same        parts or epitopes of said antigen or against two or more        different parts or epitopes of said antigen; and/or:    -   may be directed against the different antigens; or a combination        thereof.

Thus, a bivalent polypeptide of the invention for example:

-   -   may comprise two identical Nanobodies;    -   may comprise a first Nanobody directed against a first part or        epitope of an antigen and a second Nanobody directed against the        same part or epitope of said antigen or against another part or        epitope of said antigen;    -   or may comprise a first Nanobody directed against a first        antigen and a second Nanobody directed against a second antigen        different from said first antigen;        whereas a trivalent Polypeptide of the Invention for example:    -   may comprises three identical or different Nanobodies directed        against the same or different parts or epitopes of the same        antigen;    -   may comprise two identical or different Nanobodies directed        against the same or different parts or epitopes on a first        antigen and a third Nanobody directed against a second antigen        different from said first antigen; or    -   may comprise a first Nanobody directed against a first antigen,        a second Nanobody directed against a second antigen different        from said first antigen, and a third Nanobody directed against a        third antigen different from said first and second antigen,

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen and atleast one Nanobody is directed against a second Nanobody different fromthe first antigen, will also be referred to as “multispecific”Nanobodies. Thus, a “bispecific” Nanobody is a Nanobody that comprisesat least one Nanobody directed against a first antigen and at least onefurther Nanobody directed against a second antigen, whereas a“trispecific” Nanobody is a Nanobody that comprises at least oneNanobody directed against a first antigen, at least one further Nanobodydirected against a second antigen, and at least one further Nanobodydirected against a third antigen; etc.

Accordingly, in their simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against a first antigenand a second Nanobody directed against a second antigen, in which saidfirst and second Nanobody may optionally be linked via a linker sequence(as defined herein); whereas a trispecific polypeptide of the inventionin its simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first Nanobody directed against a firstantigen, a second Nanobody directed against a second antigen and a thirdNanobody directed against a third antigen, in which said first, secondand third Nanobody may optionally be linked via one or more, and inparticular one and more in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise any number of Nanobodiesdirected against two or more different antigens.

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, as well as to EP 0 822985.

Linkers for use in multivalent and multispecific polypeptides will beclear to the skilled person, and for example include gly-ser linkers,for example of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077, hinge likeregions such as the hinge regions of naturally occurring heavy chainantibodies or similar sequences. For other suitable linkers, referenceis also made to the general background art cited above. Someparticularly preferred linkers are given in SEQ ID NO's 83 to 85, inwhich the linkers of SEQ ID NO's 84 and 85 are particularly preferred.

Linkers can also provide some functionality for the multivalent ormultispecific polypeptide. For example, linkers containing one or morecharged amino acid residues (see Table 1 above) can provide improvedhydrophilic properties, whereas linkers that form or contain smallepitopes or tags can be used for the purposes of detection,identification and/or purification.

As also further described herein, a multispecific polypeptide of theinvention directed against a desired antigen and against at least oneserum protein, such as the serum proteins mentioned hereinbelow, and inparticular against human serum albumin, may show increased half-life inserum, compared to the corresponding monovalent Nanobody.

As mentioned hereinabove, the methods described herein are particularlysuited for generating such multivalent of multispecific polypeptides ofthe invention.

In a polypeptide of the invention, the at least one Nanobody may also belinked to a conventional V_(H) domain or to a natural or syntheticanalog of a V_(H) domain, optionally via a linker sequence.

In a polypeptide of the invention, the at least one Nanobody may also belinked to a V_(L) domain or to a natural or synthetic analog of a V_(L)domain, optionally via a linker sequence, so as to provide a polypeptideof the invention that is in the form analogous to a conventional scFvfragment, but containing a Nanobody instead of a V_(H) domain.

In a polypeptide of the invention, the at least one Nanobody may also belinked to one or more of a CH1, CH2 and/or CH3 domain, optionally via alinker sequence. For instance, a Nanobody linked to a suitable CH1domain could for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody. Such fragments may also be heterospecificor bispecific, i.e. directed against two or more antigens. A Nanobodylinked to suitable CH2 and CH3 domains, for example derived fromCamelids, could be used to form a monospecific or bispecific heavy chainantibody. Finally, a Nanobody linked to suitable CH1, CH2 and CH3domains, for example derived from a human being, could be used—togetherwith suitable light chains—to form an antibody that is analogous to aconventional 4-chain antibody, but in which one or both of theconventional V_(H) domains have been replaced by a Nanobody.

Also, in addition to the one or more Nanobodies, Polypeptides of theInvention can also contain functional groups, moieties or residues, forexample therapeutically active substances, such as those mentionedbelow, and/or markers or labels, such as fluorescent markers, isotopes,etc., as further described hereinbelow.

The Nanobodies of the invention, the polypeptides of the invention, andnucleic acids encoding the same, can be prepared in a manner known perse, and will be clear to the skilled person from the further descriptionherein. Some preferred, but non-limiting methods for preparing theNanobodies, polypeptides and nucleic acids include the methods andtechniques mentioned above and/or further described hereinbelow.

As will be clear to the skilled person, one particularly useful methodfor preparing a Nanobody and/or a polypeptide of the invention generallycomprises the steps of:

-   -   the expression, in a suitable host cell or host organism (also        referred to herein as a “host of the invention”) or in another        suitable expression system of a nucleic acid that encodes said        Nanobody or polypeptide of the invention (also referred to        herein as a “nucleic acid of the invention”), optionally        followed by:    -   isolating and/or purifying the Nanobody or polypeptide of the        invention thus obtained.

In particular, such a method may comprise the steps of:

-   -   cultivating and/or maintaining a host of the invention under        conditions that are such that said host of the invention        expresses and/or produces at least one Nanobody and/or        polypeptide of the invention; optionally followed by:    -   isolating and/or purifying the Nanobody or polypeptide of the        invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one embodiment of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined hereinabove.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring GPCR as a template. These and other techniques willbe clear to the skilled person, and reference is again made to thestandard handbooks, such as Sambrook et al. and Ausubel et al.,mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art. Such genetic constructs generally comprise at leastone nucleic acid of the invention that is optionally linked to one ormore elements of genetic constructs known per se, such as for exampleone or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to hereinbelow. Such genetic constructscomprising at least one nucleic acid of the invention will also bereferred to herein as “genetic constricts of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable independentreplication, maintenance and/or inheritance in the intended hostorganism. For instance, the genetic constructs of the invention may bein the form of a vector, such as for example a plasmid, cosmid, YAC, aviral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting embodiment, a genetic construct of theinvention comprises

-   a) at least one nucleic acid of the invention; operably connected to-   b) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;-   c) and optionally also-   d) one or more further elements of genetic constructs known per se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art (as    further described below); and in which said “further elements”    present in the genetic constructs may for example be 3′- or 5′-UTR    sequences, leader sequences, selection markers, expression    markers/reporter genes, and/or elements that may facilitate or    increase (the efficiency of) transformation or integration. These    and other suitable elements for such genetic constructs will be    clear to the skilled person, and may for instance depend upon the    type of construct used, the intended host cell or host organism; the    manner in which the nucleotide sequences of the invention of    interest are to be expressed (e.g. via constitutive, transient or    inducible expression); and/or the transformation technique to be    used.

Preferably, in the genetic constructs of the invention, said at leastone nucleic acid of the invention and said regulatory elements, andoptionally said one or more further elements, are “operably linked” toeach other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of” said promoter). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually also be essentially contiguous, although this may also not berequired.

Preferably, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein).

Some particularly preferred promoters include, but are not limited to,promoters known per se for the expression in bacterial cells, such asthose mentioned hereinbelow and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriateselection conditions—host cells and/or host organisms that have been(successfully) transformed with the nucleotide sequence of the inventionto be distinguished from host cells/organisms that have not been(successfully) transformed. Some preferred, but non-limiting examples ofsuch markers are genes that provide resistance against antibiotics (suchas kanamycine or ampicilline), genes that provide for temperatureresistance, or genes that allow the host cell or host organism to bemaintained in the absence of certain factors, compounds and/or (food)components in the medium that are essential for survival of thenon-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or hostorganism—it allows for the desired post-translational modificationsand/or such that it directs the transcribed mRNA to a desired part ororganelle of a cell. A leader sequence may also allow for secretion ofthe expression product from said cell. As such, the leader sequence maybe any pro-, pre-, or prepro-sequence operable in the host cell or hostorganism. Leader sequences may not be required for expression in abacterial cell.

An expression marker or reporter gene should be such that—in the hostcell or host organism—it allows for detection of the expression of (agene or nucleotide sequence present on) the genetic construct. Anexpression marker may optionally also allow for the localisation of theexpressed product, e.g. in a specific part or organelle of a cell and/orin (a) specific cell(s), tissue(s), organ(s) or part(s) of amulticellular organism. Such reporter genes may also be expressed as aprotein fusion with the amino acid sequence of the invention. Somepreferred, but non-limiting examples include fluorescent proteins suchas GFP.

Some preferred, but non-limiting examples of suitable promoters,terminator and further elements include those used in the Examplesbelow. For some (further) non-limiting examples of the promoters,selection markers, leader sequences, expression markers and furtherelements that may be present/used in the genetic constructs of theinvention—such as terminators, transcriptional and/or translationalenhancers and/or integration factors—reference is made to the generalhandbooks such as Sambrook et al. and Ausubel et al. mentioned above, aswell as to the examples that are given in WO 95/07463, WO 96/23810, WO95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO98/21355, U.S. Pat. No. 6,207,410, U.S. Pat. No. 5,693,492 and EP 1 085089. Other examples will be clear to the skilled person. Reference isalso made to the general background art cited above and the furtherreferences cited hereinbelow.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained byinserting a nucleotide sequence of the invention in a suitable(expression) vector known per se. Some preferred, but non-limitingexamples of suitable expression vectors are those used in the Examplesbelow, as well as those mentioned below.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the Nanobody or polypeptide of theinvention. Suitable hosts or host cells will be clear to the skilledperson, and may for example be any suitable fungal, prokaryotic oreukaryotic cell or cell line or any suitable fungal, prokaryotic oreukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative        strains such as strains of Escherichia coli; of Proteus, for        example of Proteus mirabilis; of Pseudomonas, for example of        Pseudomonas fluorescens; and gram-positive strains such as        strains of Bacillus, for example of Bacillus subtilis or of        Bacillus brevis; of Streptomyces, for example of Streptomyces        lividans; of Staphylococcus, for example of Staphylococcus        carnosus; and of Lactococcus, for example of Lactococcus lactis;    -   a fungal cell, including but not limited to cells from species        of Trichoderma, for example from Trichoderma reesei; of        Neurospora, for example from Neurospora crassa; of Sordaria, for        example from Sordaria macrospora; of Aspergillus, for example        from Aspergillus niger or from Aspergillus sojae; or from other        filamentous fungi;    -   a yeast cell, including but not limited to cells from species of        Saccharomyces, for example of Saccharomyces cerevisiae; of        Schizosaccharomyces, for example of Schizosaccharomyces pombe;        of Pichia, for example of Pichia pastoris or of Pichia        methanolica; of Hansenula, for example of Hansenula polymorpha;        of Kluyveromyces, for example of Kluyveromyces lactis; of        Arxula, for example of Arxula adeninivorans; of Yarrowia, for        example of Yarrowia lipolytica;    -   an amphibian cell or cell line, such as Xenopus oocytes;    -   an insect-derived cell or cell line, such as cells/cell lines        derived from lepidoptera, including but not limited to        Spodoptera SF9 and Sf21 cells or cells/cell lines derived from        Drosophila, such as Schneider and Kc cells;    -   a plant or plant cell, for example in tobacco plants; and/or    -   a mammalian cell or cell line, for example derived a cell or        cell line derived from a human, from the mammals including but        not limited to CHO-cells, BHK-cells (for example BHK-21 cells)        and human cells or cell lines such as HeLa, COS (for example        COS-7) and PER.C6 cells;        as well as all other hosts or host cells known per se for the        expression and production of antibodies and antibody fragments        (including but not limited to (single) domain antibodies and        ScFv fragments), which will be clear to the skilled person.        Reference is also made to the general background art cited        hereinabove, as well as to for example WO 94/29457; WO 96/34103;        WO 99/42077; Frenken et al., (1998), supra; Riechmann and        Muyldermans, (1999), supra; van der Linden, (2000), supra;        Thomassen et al., (2002), supra; Joosten et al., (2003), supra;        Joosten et al., (2005), supra; and the further references cited        herein.

The Nanobodies and polypeptides of the invention can also be introducedand expressed in one or more cells, tissues or organs of a multicellularorganism, for example for prophylactic and/or therapeutic purposes (e.g.as a gene therapy). For this purpose, the nucleotide sequences of theinvention may be introduced into the cells or tissues in any suitableway, for example as such (e.g. using liposomes) or after they have beeninserted into a suitable gene therapy vector (for example derived fromretroviruses such as adenovirus, or parvoviruses such asadeno-associated virus). As will also be clear to the skilled person,such gene therapy may be performed in vivo and/or in situ in the body ofa patent by administering a nucleic acid of the invention or a suitablegene therapy vector encoding the same to the patient or to specificcells or a specific tissue or organ of the patient; or suitable cells(often taken from the body of the patient to be treated, such asexplanted lymphocytes, bone marrow aspirates or tissue biopsies) may betreated in vitro with a nucleotide sequence of the invention and then besuitably (re-)introduced into the body of the patient. All this can beperformed using gene therapy vectors, techniques and delivery systemswhich are well known to the skilled person, for Culver, K. W., “GeneTherapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York,N.Y.). Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ.Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma,Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser,Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36;Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.: 811(1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang,Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat.No. 5,580,859; 1 U.S. Pat. No. 5,589,5466; or Schaper, Current Opinionin Biotechnology 7 (1996), 635-640. For example, in situ expression ofScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) andof diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has beendescribed in the art.

For expression of the Nanobodies in a cell, they may also be expressedas so-called or as so-called “intrabodies”, as for example described inWO 94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170.

For production, the Nanobodies and polypeptides of the invention can forexample also be produced in the milk of transgenic mammals, for examplein the milk of rabbits, cows, goats or sheep (see for example U.S. Pat.No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No. 6,849,992 forgeneral techniques for introducing transgenes into mammals), in plantsor parts of plants including but not limited to their leaves, flowers,fruits, seed, roots or turbers (for example in tobacco, maize, soybeanor alfalfa) or in for example pupae of the silkworm Bombix mori.

Furthermore, the Nanobodies and polypeptides of the invention can alsobe expressed and/or produced in cell-free expression systems, andsuitable examples of such systems will be clear to the skilled person.Some preferred, but non-limiting examples include expression in thewheat germ system; in rabbit reticulocyte lysates; or in the E. coliZubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,Polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceuticalexpression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast are usually leads to a glycosylation pattern that differsfrom human glycosylation. Nevertheless, it should be understood that allthe foregoing host cells and expression systems can be used in theinvention, depending on the desired Nanobody or protein to be obtained.

Thus, according to one non-limiting embodiment of the invention, theNanobody or polypeptide of the invention is glycosylated. According toanother non-limiting embodiment of the invention, the Nanobody orpolypeptide of the invention is non-glycosylated.

According to one preferred, but non-limiting embodiment of theinvention, the Nanobody or polypeptide of the invention is produced in abacterial cell, in particular a bacterial cell suitable for large scalepharmaceutical production, such as cells of the strains mentioned above.

According to another preferred, but non-limiting embodiment of theinvention, the Nanobody or polypeptide of the invention is produced in ayeast cell, in particular a yeast cell suitable for large scalepharmaceutical production, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting embodiment of theinvention, the Nanobody or polypeptide of the invention is produced in amammalian cell, in particular in a human cell or in a cell of a humancell line, and more in particular in a human cell or in a cell of ahuman cell line that is suitable for large scale pharmaceuticalproduction, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the Nanobodies and theproteins of the invention, the Nanobodies and proteins of the inventioncan be produced either intracellullarly (e.g. in the cytosol, in theperiplasma or in inclusion bodies) and then isolated from the host cellsand optionally further purified; or can be produced extracellularly(e.g. in the medium in which the host cells are cultured) and thenisolated from the culture medium and optionally further purified. Wheneukaryotic hosts cells are used, extracellular production is usuallypreferred since this considerably facilitates the further isolation anddownstream processing of the Nanobodies and proteins obtained. Bacterialcells such as the strains of E. coli mentioned above normally do notsecrete proteins extracellularly, except for a few classes of proteinssuch as toxins and hemolysin, and secretory production in E. coli refersto the translocation of proteins across the inner membrane to theperiplasmic space. Periplasmic production provides several advantagesover cytosolic production. For example, the N-terminal amino acidsequence of the secreted product can be identical to the natural geneproduct after cleavage of the secretion signal sequence by a specificsignal peptidase. Also, there appears to be much less protease activityin the periplasm than in the cytoplasm. In addition, proteinpurification is simpler due to fewer contaminating proteins in theperiplasm. Another advantage is that correct disulfide bonds may formbecause the periplasm provides a more oxidative environment than thecytoplasm. Proteins overexpressed in E. coli are often found ininsoluble aggregates, so-called inclusion bodies. These inclusion bodiesmay be located in the cytosol or in the periplasm; the recovery ofbiologically active proteins from these inclusion bodies requires adenaturation/refolding process. Many recombinant proteins, includingtherapeutic proteins, are recovered from inclusion bodies.Alternatively, as will be clear to the skilled person, recombinantstrains of bacteria that have been genetically modified so as to secretea desired protein, and in particular a Nanobody or a polypeptide of theinvention, can be used.

Thus, according to one non-limiting embodiment of the invention, theNanobody or polypeptide of the invention is a Nanobody or polypeptidethat has been produced intracellularly and that has been isolated fromthe host cell, and in particular from a bacterial cell or from aninclusion body in a bacterial cell. According to another non-limitingembodiment of the invention, the Nanobody or polypeptide of theinvention is a Nanobody or polypeptide that has been producedextracellularly, and that has been isolated from the medium in which thehost cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude,

-   -   for expression in E. coli: lac promoter (and derivatives thereof        such as the lacUV5 promoter); arabinose promoter; left- (PL) and        rightward (PR) promoter of phage lambda; promoter of the trp        operon; hybrid lac/trp promoters (tac and trc); T7-promoter        (more specifically that of T7-phage gene 10) and other T-phage        promoters; promoter of the Tn10 tetracycline resistance gene;        engineered variants of the above promoters that include one or        more copies of an extraneous regulatory operator sequence;    -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol        dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),        GAPDH (glyceraldehydes-3-phosphate dehydrogenase); PGK1        (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:        GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol        dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper        metallothionein); heterologous: CaMV (cauliflower mosaic virus        35S promoter);    -   for expression in Pichia pastoris: the AOX1 promoter (alcohol        oxidase I)    -   for expression in mammalian cells: human cytomegalovirus (hCMV)        immediate early enhancer/promoter; human cytomegalovirus (hCMV)        immediate early promoter variant that contains two tetracycline        operator sequences such that the promoter can be regulated by        the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)        promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)        enhancer/promoter; elongation factor 1α (hEF-1α) promoter from        human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1        long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cellsinclude:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),        pcDNA3 (Invitrogen), pMClneo (Stratagene), pSG5 (Stratagene),        EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),        pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo        (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and        1ZD35 (ATCC 37565), as well as viral-based expression systems,        such as those based on adenovirus;    -   vectors for expression in bacterials cells: pET vectors        (Novagen) and pQE vectors (Qiagen);    -   vectors for expression in yeast or other fungal cells: pYES2        (Invitrogen) and Pichia expression vectors (Invitrogen);    -   vectors for expression in insect cells: pBlueBacII (Invitrogen)        and other baculovirus vectors    -   vectors for expression in plants or plant cells: for example        vectors based on cauliflower mosaic virus or tobacco mosaic        virus, suitable strains of Agrobacterium, or Ti-plasmid based        vectors.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,        OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the        like; TAT signal peptide, hemolysin C-terminal secretion signal    -   for use in yeast: α-mating factor prepro-sequence, phosphatase        (phoI), invertase (Suc), etc.;    -   for use in mammalian cells: indigenous signal in case the target        protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal        peptide; etc.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence of the invention (and in case of ahost organism: in at least one cell, part, tissue or organ thereof). Theinvention also includes further generations, progeny and/or offspring ofthe host cell or host organism of the invention, that may for instancebe obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence of the invention isexpressed/produced. Suitable conditions will be clear to the skilledperson and will usually depend upon the host cell/host organism used, aswell as on the regulatory elements that control the expression of the(relevant) nucleotide sequence of the invention. Again, reference ismade to the handbooks and patent applications mentioned above in theparagraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequenceof the invention may (first) be generated in an immature form (asmentioned above), which may then be subjected to post-translationalmodification, depending on the host cell/host organism used. Also, theamino acid sequence of the invention may be glycosylated, againdepending on the host cell/host organism used.

The amino acid sequence of the invention may then be isolated from thehost cell/host organism and/or from the medium in which said host cellor host organism was cultivated, using protein isolation and/orpurification techniques known per se, such as (preparative)chromatography and/or electrophoresis techniques, differentialprecipitation techniques, affinity techniques (e.g. using a specific,cleavable amino acid sequence fused with the amino acid sequence of theinvention) and/or preparative immunological techniques (i.e. usingantibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation comprising at least onepolypeptide of the invention and at least one pharmaceuticallyacceptable carrier, diluent or excipient and/or adjuvant, and optionallyone or more further pharmaceutically active polypeptides and/orcompounds. By means of non-limiting examples, such a formulation may bein a form suitable for oral administration, for parenteraladministration (such as by intravenous, intramuscular or subcutaneousinjection or intravenous infusion), for topical administration, foradministration by inhalation, by a skin patch, by an implant, by asuppository, etc. Such suitable administration forms—which may be solid,semi-solid or liquid, depending on the manner of administration—as wellas methods and carriers for use in the preparation thereof, will beclear to the skilled person, and are further described hereinbelow.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one Nanobody of the invention or atleast one polypeptide of the invention and at least one suitable carrier(i.e. a carrier suitable for veterinary use), and optionally one or morefurther active substances.

One embodiment of the present invention is a polypeptide constructcomprising: at least one Nanobody of the invention, i.e. directedagainst any of vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3domain.

Another embodiment of the present invention is a polypeptide constructas described above, wherein the Nanobody of the invention directedagainst the A1 domain of activated vWF specifically recognizes theactivated vWF conformation at the site of thrombus formation but doesnot bind to circulating unactivated forms of vWF.

The Nanobodies of the invention may also be directed against a fragmentof vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, suchas a fragment capable of eliciting an immune response. A target is alsoa fragment of vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3domain, capable of binding to a Nanobody of the invention raised againstthe ‘parent’ full length target.

A fragment as used herein refers to less than 100% of the sequence(e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), butcomprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or more amino acids. A fragment is of sufficient lengthsuch that the interaction of interest is maintained with affinity of1×10-6 M or better.

A fragment as used herein also refers to optional insertions, deletionsand substitutions of one or more amino acids which do not substantiallyalter the ability of the target to bind to a Nanobody of the inventionraised against the wild-type target. The number of amino acid insertionsdeletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

A Nanobody of the invention directed against a target generally meansNanobody of the invention that it is capable of binding to its targetwith an affinity of better than 10⁻⁶ M.

Another embodiment of the present invention is a polypeptide constructas described above wherein at least one Nanobody of the invention is ahumanised sequence.

Another embodiment of the present invention is a polypeptide constructas described above wherein at least one Nanobody of the invention is aCamelidae V_(HH) antibody.

Another embodiment of the present invention is a polypeptide constructas described above, wherein said Nanobody of the invention is anhomologous sequence, a functional portion, or a functional portion of anhomologous sequence of the full length Nanobody of the invention.

Another embodiment of the present invention is a polypeptide constructas described above, wherein said polypeptide construct is a homologoussequence of said polypeptide construct, a functional portion thereof, ofan homologous sequence of a functional portion thereof.

Another embodiment of the present invention is a polypeptide constructas described above, further comprising at least one Nanobody of theinvention directed against one or more serum proteins, in particular oneor more human serum proteins.

Another embodiment of the present invention is a polypeptide constructas described above wherein said at least one (human) serum protein isany of (human) serum albumin, (human) serum immunoglobulins, (human)thyroxine-binding protein, (human) transferrine, or (human) fibrinogenor a fragment thereof.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNanobodies of the invention, at least one Nanobody against human serumalbumin. Although these Nanobodies against human serum albumin may be asgenerally described in W04/062551 or in the further references citedtherein, according to a particularly preferred, but non-limitingembodiment, said Nanobody against human serum albumin consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

-   i) CDR1 is an amino acid sequence chosen from the group consisting    of:

SFGMS [SEQ ID NO: 44] LNLMG [SEQ ID NO: 45] INLLG [SEQ ID NO: 46] NYWMY;[SEQ ID NO: 47]

-   -   and/or from the group consisting of amino acid sequences that        have 2 or only 1 “amino acid difference(s)” (as defined herein)        with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequences;            and in which:

-   ii) CDR2 is an amino acid sequence chosen from the group consisting    of:

SISGSGSDTLYADSVKG [SEQ ID NO: 48] TITVGDSTNYADSVKG [SEQ ID NO: 49]TITVGDSTSYADSVKG [SEQ ID NO: 50] SINGRGDDTRYADSVKG [SEQ ID NO: 51]AISADSSTKNYADSVKG [SEQ ID NO: 52] AISADSSDKRYADSVKG [SEQ ID NO: 53]RISTGGGYSYYADSVKG [SEQ ID NO: 54]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequences;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequences;            and in which:

-   iii) CDR3 is an amino acid sequence chosen from the group consisting    of:

DREAQVDTLDFDY [SEQ ID NO: 55]

-   -   or from the group consisting of amino acid sequences that have        at least 80%, preferably at least 90%, more preferably at least        95%, even more preferably at least 99% sequence identity (as        defined herein) with one of the above amino acid sequences; in        which        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequences;    -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequences;            or from the group consisting of:

GGSLSR [SEQ ID NO: 56] RRTWHSEL [SEQ ID NO: 57] GRSVSRS [SEQ ID NO: 58]GRGSP [SEQ ID NO: 59]

-   -   and/or from the group consisting of amino acid sequences that        have 3, 2 or only 1 “amino acid difference(s)” (as defined        herein) with one of the above amino acid sequences, in which:        -   (1) any amino acid substitution is preferably a conservative            amino acid substitution (as defined herein); and/or        -   (2) said amino acid sequence preferably only contains amino            acid substitutions, and no amino acid deletions or            insertions, compared to the above amino acid sequences.

In another aspect, the invention relates to a Nanobody against vWF,which consist of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), whichis chosen from the group consisting of Nanobodies with the one of thefollowing combinations of CDR1, CDR2 and CDR3, respectively:

CDR1: SFGMS [SEQ ID NO: 44]; CDR2: SISGSGSDTLYADSVKG [SEQ ID NO: 48];CDR3: GGSLSR [SEQ ID NO: 56]; CDR1: LNLMG [SEQ ID NO: 45]; CDR2:TITVGDSTNYADSVKG [SEQ ID NO: 49]; CDR3: RRTWHSEL [SEQ ID NO: 57]; CDR1:INLLG [SEQ ID NO: 46]; CDR2: TITVGDSTSYADSVKG [SEQ ID NO: 50]; CDR3:RRTWHSEL [SEQ ID NO: 57]; CDR1: SFGMS [SEQ ID NO: 44]; CDR2:SINGRGDDTRYADSVKG [SEQ ID NO: 51]; CDR3: GRSVSRS [SEQ ID NO: 58]; CDR1:SFGMS [SEQ ID NO: 44]; CDR2: AISADSSDKRYADSVKG [SEQ ID NO: 53]; CDR3:GRGSP [SEQ ID NO: 59]; CDR1: SFGMS [SEQ ID NO: 44]; CDR2:AISADSSDKRYADSVKG [SEQ ID NO: 53]; CDR3: GRGSP [SEQ ID NO: 59]; CDR1:NYWMY [SEQ ID NO: 47]; CDR2: RISTGGGYSYYADSVKG [SEQ ID NO: 54]; CDR3:DREAQVDTLDFDY [SEQ ID NO: 55].

In the Nanobodies of the invention that comprise the combinations ofCDR's mentioned above, each CDR can be replaced by a CDR chosen from thegroup consisting of amino acid sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with thementioned CDR's; in which

-   -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences;        and/or chosen from the group consisting of amino acid sequences        that have 3, 2 or only 1 (as indicated in the preceding        paragraph) “amino acid difference(s)” (as defined herein) with        the mentioned CDR(s) one of the above amino acid sequences, in        which:    -   (1) any amino acid substitution is preferably a conservative        amino acid substitution (as defined herein); and/or    -   (2) said amino acid sequence preferably only contains amino acid        substitutions, and no amino acid deletions or insertions,        compared to the above amino acid sequences.

However, of the Nanobodies of the invention that comprise thecombinations of CDR's mentioned above, Nanobodies comprising one or moreof the CDR's listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's listed above are more particularlypreferred; and Nanobodies comprising three of the CDR's listed above aremost particularly preferred.

In these Nanobodies against human serum albumin, the Framework regionsFR1 to FR4 are preferably as defined hereinabove for the Nanobodies ofthe invention.

Particularly preferred Nanobodies against human serum albumin are chosenfrom the group consisting of SEQ ID NO's: 107-121. These correspond tothe Nanobodies against human serum albumin of SEQ ID NO's: 61 to 67, SEQID NO's 87 to 89 and SEQ ID NO's 100-104 from applicant's co-pending USprovisional application entitled “Improved Nanobodies™ against TumorNecrosis Factor-alpha” with a filing date of May 18, 2005.

More generally, Nanobodies against serum albumin suitable for use in theinvention are described in the International application by applicantentitled “serum albumin binding proteins” with an international filingdate of May 17, 2006.

Another embodiment of the present invention is a nucleic acid encoding apolypeptide construct as described above.

Another embodiment of the present invention is a composition comprisinga polypeptide construct as described above and at least one thrombolyticagent, for simultaneous, separate or sequential administration to asubject.

Another embodiment of the present invention is a composition asdescribed above wherein said thrombolytic agent is any ofstaphylokinase, tissue plasminogen activator, streptokinase, singlechain streptokinase, urokinase and acyl plasminogen streptokinasecomplex.

Another embodiment of the present invention is a polypeptide constructas described above, or a nucleic acid as described above, or acomposition as described above for use in the treatment, preventionand/or alleviation of disorders relating to platelet-mediate aggregationor dysfunction thereof.

Another embodiment of the present invention is a use of a polypeptideconstruct as described above, or a nucleic acid as described above, or acomposition as described above for the preparation of a medicament forthe treatment, prevention and/or alleviation of disorders relating toplatelet-mediate aggregation or dysfunction thereof.

Another embodiment of the present invention is a polypeptide construct,nucleic acid or composition as described above or a use of a polypeptideconstruct, nucleic acid or composition as described above wherein saiddisorders are any arising from transient cerebral ischemic attack,unstable or stable angina, angina pectoris, cerebral infarction,myocardial infarction, peripheral arterial occlusive disease,restenosis, coronary by-pass graft, or coronary artery valve replacementand coronary interventions such angioplasty, stenting, carotidendarterectomy or atherectomy.

Another embodiment of the present invention is a polypeptide construct,nucleic acid or composition as described above or a use of a polypeptideconstruct, nucleic acid or composition as described above wherein saiddisorders are any of the formation of a non-occlusive thrombus, theformation of an occlusive thrombus, arterial thrombus formation, acutecoronary occlusion, restenosis, restenosis after PCTA or stenting,thrombus formation in stenosed arteries, hyperplasia after angioplasty,atherectomy or arterial stenting, occlusive syndrome in a vascularsystem or lack of patency of diseased arteries.

Another embodiment of the present invention is a polypeptide construct,nucleic acid or composition as described above or a use of a polypeptideconstruct, nucleic acid or composition as described above wherein saiddisorder is plaque or thrombus formation in high sheer environments.

Another embodiment of the present invention is a polypeptide construct,nucleic acid or composition as described above or a use of a polypeptideconstruct as described above wherein said polypeptide construct isadministered intravenously, subcutaneously, orally, sublingually,topically, nasally, vaginally, rectally or by inhalation.

Another embodiment of the present invention is a composition comprisinga polypeptide construct as described above or a nucleic acid encodingsaid polypeptide construct, or a composition as described above and apharmaceutically acceptable vehicle.

Another embodiment of the present invention is a method of producing apolypeptide as described above, comprising

(a) culturing host cells comprising nucleic acid capable of encoding apolypeptide as described above under conditions allowing the expressionof the polypeptide, and,

(b) recovering the produced polypeptide from the culture.

Another embodiment of the present invention is a method as describedabove, wherein said host cells are bacterial or yeast.

Another embodiment of the present invention is a method for treatinginvasive medical devices to prevent platelet-mediate aggregation aroundthe site of invasion comprising the step of coating said device with apolypeptide construct as described above.

Another embodiment of the present invention is an invasive medicaldevice for circumventing platelet-mediate aggregation around the site ofinvasion, wherein said device is coated with a polypeptide construct asdescribed above.

Another embodiment of the present invention is a method of identifyingan agent that modulates platelet-mediated aggregation comprising

(a) contacting a polypeptide construct as described above with apolypeptide corresponding to its target, or a fragment thereof, in thepresence and absence of a candidate modulator under conditionspermitting binding between said polypeptides, and

(b) measuring the binding between the polypeptides of step (a), whereina decrease in binding in the presence of said candidate modulator,relative to the binding in the absence of said candidate modulatoridentified said candidate modulator as an agent that modulateplatelet-mediated aggregation.

Another embodiment of the present invention is a kit for screening foragents that modulate platelet-mediated aggregation according to themethod as described above.

Another embodiment of the present invention is an unknown agent thatmodulates platelet-mediated aggregation identified according to themethod as described above.

Another embodiment of the present invention is a method of diagnosing adisease or disorder characterised by dysfunction of platelet-mediatedaggregation comprising the steps of:

(a) contacting a sample with a polypeptide construct as described above,and

(b) detecting binding of said polypeptide construct to said sample, and

(c) comparing the binding detected in step (b) with a standard, whereina difference in binding relative to said sample is diagnostic of adisease or disorder characterised by dysfunction of platelet-mediatedaggregation.

Another embodiment of the present invention is a kit for screening fordiagnosing a disease or disorder characterised by dysfunction ofplatelet-mediated aggregation according to the method as describedabove.

Another embodiment of the present invention is a kit as described abovecomprising a polypeptide construct as described above.

In the polypeptides of the invention, the one or more Nanobodies of theinvention which are directed against a target may be of the samesequence. Alternatively they may not all have the same sequence. It iswithin the scope of the invention that a polypeptide of the inventioncomprises anti-target Nanobodies of the invention which do not all sharethe same sequence, but which are directed against the same target, orfragment thereof, one or more antigens thereof.

It is another aspect of the invention that the polypeptide of theinvention comprises two or more Nanobodies of the invention, wherein anytwo Nanobodies of the invention are directed against differentepitopes/targets, i.e. against any of vWF, vWF A1 domain, A1 domain ofactivated vWF, vWF A3 domain.

Another aspect of the invention is a bispecific polypeptide of theinvention comprising a Nanobody of the invention directed against vWF A1domain, A1 domain of activated vWF, and another Nanobody of theinvention directed against vWF A3 domain. Said bispecific polypeptide ofthe invention inhibits the interaction between vWF and collagen, and theinteraction between vWF and platelets.

According to an aspect of the present invention a polypeptide of theinvention may comprise two or more Nanobodies of the invention whichhave been joined. The Nanobodies of the invention may be identical insequence and directed against the same target or antigen. Depending onthe number of V_(HH)s linked, a multivalent V_(HH) may be bivalent (2V_(HH)s), trivalent (3 V_(HH)s), tetravalent (4 V_(HH)s) or have ahigher valency molecules.

The present invention also relates to the finding that a polypeptide ofthe invention further comprising one or more Nanobodies of the inventioneach directed against a serum protein of a subject, surprisingly hassignificantly prolonged half-life in the circulation of said subjectcompared with the half-life of the anti-target Nanobody of theinvention(ies) when not part of said construct. Furthermore, the saidconstructs were found to exhibit the same favourable properties ofV_(HH)s such as high stability remaining intact in mice, extreme pHresistance, high temperature stability and high target affinity.

The serum protein may be any suitable protein found in the serum ofsubject, or fragment thereof. In one aspect of the invention, the serumprotein is serum albumin, serum immunoglobulins, thyroxine-bindingprotein, transferrin, or fibrinogen. Depending on the intended use suchas the required half-life for effective treatment and/orcompartimentalisation of the target antigen, the V_(HH)-partner can bedirected to one of the above serum proteins.

Such constructs are able to circulate in the subject's serum for severaldays, reducing the frequency of treatment, the inconvenience to thesubject and resulting in a decreased cost of treatment. Furthermore, itis an aspect of the invention that the half-life of the polypeptide ofthe invention disclosed herein may be controlled by the number ofanti-serum protein Nanobodies of the invention present in the construct.A controllable half-life is desirable in several circumstances, forexample, in the application of a timed dose of a therapeutic polypeptideof the invention.

Another embodiment of the present invention is a polypeptide of theinvention as mentioned herein, further comprising a thrombolytic agent.

Said thrombolytic agent may be non-covalently or covalently attached toa Nanobody of the invention via covalent or non-covalent means. Suchcovalent means are described below. Non-covalent means include via aprotein interaction such as biotin/strepavidin, or via animmunoconjugate.

Alternatively, the thrombolytic agent may be administered simultaneous,separate or sequential in respect of a polypeptide of the invention.

Another aspect of the invention is a composition comprising at least onepolypeptide of the invention and at least one thrombolytic agent, forsimultaneous, separate or sequential administration to a subject.

One aspect of the invention is a method for treating autoimmune diseasecomprising administering to an individual an effective amount of atleast one polypeptide of the invention and at least one thrombolyticagent, simultaneously, separately or sequentially.

Another aspect of the invention is a kit containing at least onepolypeptide of the invention and at least one thrombolytic agent forsimultaneous, separate or sequential administration to a subject. It isan aspect of the invention that the kit may be used according to theinvention. It is an aspect of the invention that the kit may be used totreat the diseases as cited herein.

By simultaneous administration means the polypeptide and thrombolyticagent are administered to a subject at the same time. For example, as amixture or a composition comprising said components. Examples include,but are not limited to a solution administered intravenously, a tablet,liquid, topical cream, etc., wherein each preparation comprises thecomponents of interest.

By separate administration means polypeptide and thrombolytic agent areadministered to a subject at the same time or substantially the sametime. The components are present in the kit as separate, unmixedpreparations. For example, the polypeptide and thrombolytic agent may bepresent in the kit as individual tablets. The tablets may beadministered to the subject by swallowing both tablets at the same time,or one tablet directly following the other.

By sequential administration means the polypeptide and thrombolyticagent are administered to a subject sequentially. The polypeptide andthrombolytic agent are present in the kit as separate, unmixedpreparations. There is a time interval between doses. For example, onecomponent might be administered up to 336, 312, 288, 264, 240, 216, 192,168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8, 4, 2, 1, or 0.5 hoursafter the other component.

In sequential administration, one component may be administered once, orany number of times and in various doses before and/or afteradministration of another component. Sequential administration may becombined with simultaneous or sequential administration.

The medical uses of the polypeptide of the invention described below,also apply to the composition comprising a polypeptide of the inventionand at least one polypeptide thrombolytic agent, for simultaneous,separate or sequential administration to a subject as disclosed hereabove.

Thrombolytic agents according to the invention may include, for example,staphylokinase, tissue plasminogen activator, streptokinase, singlechain streptokinase, urokinase and acyl plasminogen streptokinasecomplex.

The Nanobodies of the invention may be joined to form any of thepolypeptide of the invention disclosed herein comprising more than oneNanobody of the invention using methods known in the art or any futuremethod. For example, they may be fused by chemical cross-linking byreacting amino acid residues with an organic derivatisation agent suchas described by Blattler et al, Biochemistry 24, 1517-1524; EP294703.Alternatively, the Nanobody of the invention may be fused genetically atthe DNA level i.e. a polynucleotide construct formed which encodes thecomplete polypeptide of the invention comprising one or more anti-targetNanobodies of the invention and one or more anti-serum proteinNanobodies of the invention. A method for producing bivalent ormultivalent V_(HH) polypeptide of the invention is disclosed in PCTpatent application WO 96/34103. One way of joining multiple Nanobodiesof the invention is via the genetic route by linking Nanobody of theinvention coding sequences either directly or via a peptide linker. Forexample, the C-terminal end of the first Nanobody of the invention maybe linked to the N-terminal end of the next Nanobody of the invention.This linking mode can be extended in order to link additional Nanobodiesof the invention for the construction and production of tri-, tetra-,etc. functional constructs.

The polypeptide of the invention disclosed herein may be made by theskilled artisan according to methods known in the art or any futuremethod. For example, V_(HH)s may be obtained using methods known in theart such as by immunising a camel and obtaining hybridoma's therefrom,or by cloning a library of Nanobodies of the invention using molecularbiology techniques known in the art and subsequent selection by usingphage display.

Nanobodies have a unique structure that consists of a single variabledomain. V_(HH) molecules derived from Camelidae antibodies are among thesmallest intact antigen-binding domains known (approximately 15 kDa, or10 times smaller than a conventional IgG) and hence are well suitedtowards delivery to dense tissues and for accessing the limited spacebetween macromolecules participating in or starting the process ofplatelet mediated aggregation.

Despite the small size of nanobodies, and thus advantages forpenetration, it is still surprising that such a small molecule caninhibit interactions between large polymers such as vWF (up to 60monomers) and collagen and with such a high efficiency. It has beendescribed that only the large multimeric forms of vWF are hemostaticallyactive (Furlan, M. 1996, Ann. Hematol. 72:341-348). Binding ofmultimeric vWF to collagen occurs with ˜100-fold higher affinity thanbinding of monomeric vWF fragments.

The results from the high shear experiments indicate that a lower dosemay be administered to patients. Therefore, fewer side effects areexpected (such as immunogenicity or bleeding problems).

In another embodiment of the present invention, a polypeptide of theinvention comprises one or more Nanobodies of the invention directed tothe same target, and further comprises one or more Nanobodies of theinvention directed to the same target but to a different epitope in thesame domain.

Another embodiment of the present invention is a polypeptide of theinvention wherein the number of Nanobodies of the invention directed tothe same target is two or more.

In another embodiment of the present invention, a polypeptide of theinvention comprises one or more Nanobodies of the invention directed toone domain of the same target, and one or more Nanobodies of theinvention directed to the same target but to another domain of the sametarget. Examples of different domains might be the A1 and A3 domains ofvWF

It is one non-limiting aspect of the invention that at least one V_(HH)directed to the A1 domain in a heterospecific polypeptide of theinvention recognizes the active conformation of vWF. Such polypeptide ofthe invention may have superior anti-thrombotic effects compared to themonomeric V_(HH)'s. Perfusion experiment were performed in a flowchamber, to study platelet aggregation under high shear to study theeffects of these polypeptide of the invention.

The discovery of naturally occurring Nanobodies of the invention inllama, dromedary and camel revealed a new class of therapeutic moleculeswhich combine the advantages of monoclonal antibodies for examplespecificity, low toxicity with the advantages of small molecules forexample tissue penetration and stability. Unfortunately, the developmentof appropriate therapeutic products based on these proteins has thedrawback of being Camelidae derived, and thus not human. Non-humanproteins contain amino acid residues that can be immunogenic wheninjected into a human patient. Although studies have shown thatCamelidae-derived V_(HH) are not immunogenic when injected in mice,replacing Camelidae residues by human residues is preferable. Thesehumanized polypeptides should be substantially non-immunogenic inhumans, but retain the affinity and activity of the wild typepolypeptide.

The result of humanisation is preferably that immunogenicity uponadministration in human patients is minor or nonexistent. Humanising apolypeptide, according to the present invention, comprises a step ofreplacing one or more of the Camelidae amino acids by their humancounterpart as found in the human consensus sequence, without thatpolypeptide losing its typical character, i.e. the humanisation does notsignificantly affect the antigen binding capacity of the resultingpolypeptide.

WO 04/062551 and the further description herein describe some preferred,but non-limiting examples of amino acid residues of the antibodyvariable domain (V_(HH)) which may be modified without diminishing thenative affinity of the domain for antigen and while reducing itsimmunogenicity with respect to a heterologous species; the use ofV_(HH)s having modifications at the identified residues which are usefulfor administration to heterologous species; and to the V_(HH) somodified. More specifically, the invention also encompasses thepreparation of modified V_(HH)s, which are modified for administrationto humans, the resulting V_(HH) themselves, and the use of such“humanized” V_(HH)s in the treatment of diseases in humans.

As mentioned in WO 04/062551 and in the further description herein,humanization of V_(HH) polypeptides requires the introduction andmutagenesis of only a limited number of amino acids in a singlepolypeptide chain without dramatic loss of binding and/or inhibitionactivity. This is in contrast to humanization of scFv, Fab, (Fab)2 andIgG, which requires the introduction of amino acid changes in twochains, the light and the heavy chain and the preservation of theassembly of both chains.

A humanisation technique may be performed by a method comprising thereplacement of any of the following residues either alone or incombination: FR1 positions 1, 5, 28 and 30, the hallmark amino acid atposition 37, 44, 45 and 47 in FR2, FR3 residues 74, 75, 76, 83, 84, 93and 94 and positions 103, 104, 108 and 111 in FR4; numbering accordingto the Kabat numbering.

The Nanobodies of the invention have a high degree of homology to humangermline VH DP-47. Further humanization may also involve theintroduction and mutagenesis of a limited amount of amino acids in asingle polypeptide chain. This is in contrast to humanization of scFv,Fab, (Fab)2 and IgG, which requires the introduction of amino acidchanges in two chains, the light and the heavy chain and thepreservation of the assembly of both chains.

The polypeptides contain human-like residues in FR2. Humanization mayalso involve mutagenesis of residues in FR1 at position 1 and 5 whichwere introduced by the primer used for repertoire cloning and do notoccur naturally in the llama sequence. Mutagenesis of those residues didnot result in loss of binding and/or inhibition activity. Humanizationof FR1 also required mutagenesis of position 28 and 30. Mutagenesis ofthose residues also did not result in loss of binding and/or inhibitionactivity.

Humanization may also involve mutagenesis of residues in FR3 at position74, 75, 76, 83, 84, 93, 94. Mutagenesis of those residues did not resultin loss of binding and/or inhibition activity.

Humanization may also involve mutagenesis of residues in FR4 at position104, 108 and 111. Mutagenesis of Q108L resulted in lower productionlevel in Escherichia coli. Position 108 is solvent exposed in camelidV_(HH), while in human antibodies this position is buried at the VH-VLinterface (Spinelli, 1996; Nieba, 1997). In isolated V_(H)s position 108is solvent exposed. The introduction of a non-polar hydrophobic Leuinstead of polar uncharged Gln can have a drastic effect on theintrinsic foldability/stability of the molecule.

One embodiment of the present invention is a method for humanizing aV_(HH) comprising the steps of:

(a) replacing of any of the following residues either alone or incombination:

FR1 positions 1, 5, 28 and 30,

the hallmark amino acid at position 37, 44, 45 and 47 in FR2,

FR3 residues 74, 75, 76, 83, 84, 93 and 94,

and positions 103, 104, 108 and 111 in FR4;

numbering according to the Kabat numbering.

Examples of such humanized sequences are given below and in the appendedsequence listing.

The use of antibodies derived from sources such as mouse, sheep, goat,rabbit etc., and humanised derivatives thereof as a treatment forconditions which require a modulation of platelet-associatedaggregation, is problematic for several reasons. Traditional antibodiesare not stable at room temperature, and have to be refrigerated forpreparation and storage, requiring necessary refrigerated laboratoryequipment, storage and transport, which contribute towards time andexpense. Refrigeration is sometimes not feasible in developingcountries. The yields of expression of said Fab molecules are very lowand the method of production is very labor intensive. Furthermore, themanufacture or small-scale production of said antibodies is expensivebecause the mammalian cellular systems necessary for the expression ofintact and active antibodies require high levels of support in terms oftime and equipment, and yields are very low. Furthermore, traditionalantibodies have a binding activity which depends upon pH, and hence areunsuitable for use in environments outside the usual physiological pHrange such as, for example, in treating gastric bleeding, gastricsurgery. Furthermore, traditional antibodies are unstable at low or highpH and hence are not suitable for oral administration. However, it hasbeen demonstrated that camelid antibodies resist harsh conditions, suchas extreme pH, denaturing reagents and high temperatures (Ewert S et al,Biochemistry 2002 Mar. 19; 41(11):3628-36), so making them suitable fordelivery by oral administration. Furthermore, traditional antibodieshave a binding activity which depends upon temperature, and hence areunsuitable for use in assays or kits performed at temperatures outsidebiologically active-temperature ranges (e.g. 37±20° C.).

The Nanobodies and polypeptides of the invention not only possess theadvantageous characteristics of conventional antibodies, such as lowtoxicity and high selectivity, but they also exhibit additionalproperties. They are more soluble, meaning they may be stored and/oradministered in higher concentrations compared with conventionalantibodies. They are stable at room temperature meaning they may beprepared, stored and/or transported without the use of refrigerationequipment, conveying a cost, time and environmental savings. Otheradvantageous characteristics as compared to conventional antibodiesinclude short half-life in the circulation which may be modulatedaccording to the invention by, for example, albumin-coupling, abispecific nanobody with one specificity against albumin and the otheragainst the target, Fc coupling, V_(HH) coupling (bivalent V_(HH)s) orby pegylation. A short and controllable half-life is desirable forsurgical procedures, for example, which require an inhibition ofplatelet-mediated aggregation for a limited time period. Also, whenbleeding problems occur or other complications, dosage can be loweredimmediately. The polypeptides of the present invention also retainbinding activity at a pH and temperature outside those of usualphysiological ranges, which means they may be useful in situations ofextreme pH and temperature which require a modulation ofplatelet-mediated aggregation, such as in gastric surgery, control ofgastric bleeding, assays performed at room temperature etc. Thepolypeptides of the present invention also exhibit a prolonged stabilityat extremes of pH, meaning they would be suitable for delivery by oraladministration. The polypeptides of the present invention may becost-effectively produced through fermentation in convenient recombinanthost organisms such as Escherichia coli and yeast; unlike conventionalantibodies which also require expensive mammalian cell culturefacilities, achievable levels of expression are high. Examples of yieldsof the polypeptides of the present invention are 1 to 10 mg/ml (E. coli)and up to 1 g/l (yeast). The polypeptides of the present invention alsoexhibit high binding affinity for a broad range of different antigentypes, and ability to bind to epitopes not recognised by conventionalantibodies; for example they display long CDR-based loop structures withthe potential to penetrate into cavities and exhibit enzyme functioninhibition. Furthermore, since binding often occurs through the CDR3loop only, it is envisaged that peptides derived from CDR3 could be usedtherapeutically (Desmyter et al., J Biol Chem, 2001, 276: 26285-90). Thepolypeptides of the invention are also able to retain full bindingcapacity as fusion protein with an enzyme or toxin. Furthermore, itmight be expected that the undesirable thrombocytopenia caused by Fc:Fcreceptor mediated activation of platelet aggregation and/orF(ab′)(2)-mediated crosslinking of platelets which has been observedwhen using intact IgG or F(ab′)(2) therapeutically in vivo (seeCauwenberghs N. et al, Arteriosclerosis, Thrombosis and Vascularbiology, 2000, 20: 1347), will be avoided in the use of V_(HH), sinceV_(HH) contains no Fc and it is not bivalent. Thus the polypeptides ofthe invention, homologues or functional portions thereof provide aconsiderable cost and time saving in the treatment and diagnosis ofconditions related to platelet-mediated aggregation, and the patient inneed of said polypeptides would encounter fewer of the problemsassociated with conventional agents.

Platelet-mediated aggregation is the process wherein vWF-bound collagenadheres to platelets and/or platelet receptors, ultimately resulting inplatelet activation. Platelet activation leads to fibrinogen binding,and finally to platelet aggregation. It is within the scope of thepresent invention to provide polypeptides which modulate the processeswhich comprise platelet-mediated aggregation such as vWF-collagenbinding, vWF-platelet receptor adhesion, collagen-platelet receptoradhesion, platelet activation, fibrinogen binding and/or plateletaggregation.

According to an aspect of the invention a polypeptide of the inventionmay be a homologous sequence of a full-length polypeptide of theinvention. According to another aspect of the invention, a polypeptideof the invention may be a functional portion of a full-lengthpolypeptide of the invention. According to another aspect of theinvention, a polypeptide of the invention may be a homologous sequenceof a full length polypeptide of the invention. According to anotheraspect of the invention, a polypeptide of the invention may be afunctional portion of a homologous sequence of a full length polypeptideof the invention. According to an aspect of the invention a polypeptideof the invention may comprise a sequence of a polypeptide of theinvention.

According to an aspect of the invention a Nanobody of the invention usedto form a polypeptide of the invention may be a complete Nanobody of theinvention (e.g. a V_(HH)) or a homologous sequence thereof. According toanother aspect of the invention, a Nanobody of the invention used toform the polypeptide of the invention may be a functional portion of acomplete Nanobody of the invention. According to another aspect of theinvention, a Nanobody of the invention used to form the polypeptide ofthe invention may be a homologous sequence of a complete Nanobody of theinvention. According to another aspect of the invention, a Nanobody ofthe invention used to form the polypeptide of the invention may be afunctional portion of a homologous sequence of a complete Nanobody ofthe invention.

According to another aspect of the invention a polypeptide of theinvention may be an homologous sequence of the parent sequence.According to another aspect of the invention, a polypeptide of theinvention may be a functional portion parent sequence. According toanother aspect of the invention, a polypeptide of the invention may be afunctional portion of a homologous sequence of the parent sequence.

As used herein, an homologous sequence may comprise additions, deletionsor substitutions of one or more amino acids, which do not substantiallyalter the functional characteristics of the polypeptide. The number ofamino acid deletions or substitutions is preferably up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69 or 70 amino acids.

A homologous sequence according to the present invention includespolypeptides extended by the addition of amino acids to form human heavychain antibody or human single domain heavy chain antibody, which do notsubstantially alter the functional characteristics of the unmodifiedpolypeptide.

Where homologous sequence indicates sequence identity, it means asequence which presents a high sequence identity (more than 70%, 75%,80%, 85%, 90%, 95% or 98% sequence identity) with the parent sequence,and is preferably characterised by similar properties of the parentsequence, namely affinity, said identity calculated using known methods.

Alternatively, an homologous sequence may also be any amino acidsequence resulting from allowed substitutions at any number of positionsof the parent sequence according to the formula below:

Ser substituted by Ser, Thr, Gly, and Asn;

Arg substituted by one of Arg, His, Gln, Lys, and Glu;

Leu substituted by one of Leu, Ile, Phe, Tyr, Met, and Val;

Pro substituted by one of Pro, Gly, Ala, and Thr;

Thr substituted by one of Thr, Pro, Ser, Ala, Gly, His, and Gln;

Ala substituted by one of Ala, Gly, Thr, and Pro;

Val substituted by one of Val, Met, Tyr, Phe, Ile, and Leu;

Gly substituted by one of Gly, Ala, Thr, Pro, and Ser;

Ile substituted by one of Ile, Met, Tyr, Phe, Val, and Leu;

Phe substituted by one of Phe, Trp, Met, Tyr, Ile, Val, and Leu;

Tyr substituted by one of Tyr, Trp, Met, Phe, Ile, Val, and Leu;

His substituted by one of His, Glu, Lys, Gln, Thr, and Arg;

Gln substituted by one of Gln, Glu, Lys, Asn, His, Thr, and Arg;

Asn substituted by one of Asn, Glu, Asp, Gln, and Ser;

Lys substituted by one of Lys, Glu, Gln, His, and Arg;

Asp substituted by one of Asp, Glu, and Asn;

Glu substituted by one of Glu, Asp, Lys, Asn, Gln, His, and Arg;

Met substituted by one of Met, Phe, Ile, Val, Leu, and Tyr.

A homologous according to the present invention may refer to nucleotidesequences of more than 50, 100, 200, 300, 400, 500, 600, 800 or 1000nucleotides able to hybridize to the reverse-complement of thenucleotide sequence capable of encoding a polypeptide under stringenthybridisation conditions (such as the ones described by SAMBROOK et al.,Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratorypress, New York).

As used herein, a functional portion refers to a Nanobody of theinvention of sufficient length such that the interaction of interest ismaintained with affinity of 1×10-6 M or better.

Alternatively a functional portion of a Nanobody of the inventioncomprises a partial deletion of the complete amino acid sequence andstill maintains the binding site(s) and protein domain(s) necessary forthe binding of and interaction with the target.

Alternatively a functional portion of any of Nanobody of the inventionis a polypeptide which comprises a partial deletion of the completeamino acid sequence and which still maintains the binding site(s) andprotein domain(s) necessary for the inhibition of binding of vWF tocollagen.

Alternatively a functional portion of any Nanobody of the invention is apolypeptide which comprises a partial deletion of the complete aminoacid sequence and which still maintains the binding site(s) and proteindomain(s) necessary for the binding of and interaction with the A1domain of vWF.

Alternatively a functional portion of any Nanobody of the invention is apolypeptide which comprises a partial deletion of the complete aminoacid sequence and which still maintains the binding site(s) and proteindomain(s) necessary for the binding of and interaction with collagen.

Alternatively a functional portion comprises a partial deletion of thecomplete amino acid sequence of a polypeptide and which still maintainsthe binding site(s) and protein domain(s) necessary for the binding ofand interaction with the antigen against which it was raised. Itincludes, but is not limited to V_(HH) domains.

As used herein, a functional portion as it refers to a polypeptidesequence refers to less than 100% of the sequence (e.g., 99%, 90%, 80%,70%, 60% 50% etc.), but comprising 5 or more amino acids.

A portion as it refers to a nucleotide sequence encoding a polypeptidesequence refers to less than 100% of the sequence (e.g., 99%, 90%, 80%,70%, 60% 50% etc.), but comprising 15 or more nucleotides.

An aspect of the present invention is the administration of apolypeptide of the invention according to the invention can avoid theneed for injection. Conventional antibody-based therapeutics havesignificant potential as drugs because they have exquisite specificityto their target and a low inherent toxicity, however, they have oneimportant drawback: they are relatively unstable, and are sensitive tobreakdown by proteases. This means that conventional antibody drugscannot be administered orally, sublingually, topically, nasally,vaginally, rectally or by inhalation because they are not resistant tothe low pH at these sites, the action of proteases at these sites and inthe blood and/or because of their large size. They have to beadministered by injection (intravenously, subcutaneously, etc.) toovercome some of these problems. Administration by injection requiresspecialist training in order to use a hypodermic syringe or needlecorrectly and safely. It further requires sterile equipment, a liquidformulation of the therapeutic polypeptide, vial packing of saidpolypeptide in a sterile and stable form and, of the subject, a suitablesite for entry of the needle. Furthermore, subjects commonly experiencephysical and psychological stress prior to and upon receiving aninjection.

An aspect of the present invention overcomes these problems of the priorart, by providing the polypeptides constructs of the present invention.Said constructs are sufficiently small, resistant and stable to bedelivered orally, sublingually, topically, nasally, vaginally, rectallyor by inhalation substantial without loss of activity. The polypeptidesconstructs of the present invention avoid the need for injections, arenot only cost/time savings, but are also more convenient and morecomfortable for the subject.

One embodiment of the present invention is a polypeptide of theinvention for use in treating, preventing and/or alleviating thesymptoms of disorders susceptible to modulation by a substance thatcontrols platelet mediated aggregation which is able pass through thegastric environment without the substance being inactivated.

As known by persons skilled in the art, once in possession of saidpolypeptide of the invention, formulation technology may be applied torelease a maximum amount of polypeptide in the right location (in thestomach, in the colon, etc.). This method of delivery is important fortreating, prevent and/or alleviate the symptoms of disorders whosetargets are located in the gut system.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of a disorder susceptible to modulation by asubstance that controls platelet mediated aggregation which is able passthrough the gastric environment without being inactivated, by orallyadministering to a subject a polypeptide of the invention.

Another embodiment of the present invention is a use of a Nanobody orpolypeptide of the invention for the preparation of a medicament fortreating, preventing and/or alleviating the symptoms of disorderssusceptible to modulation by a substance that controls platelet mediatedaggregation which is able pass through the gastric environment withoutbeing inactivated.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the gut system without saidsubstance being inactivated, by orally administering to a subject aNanobody or polypeptide of the invention.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the bloodstream of a subjectwithout the substance being inactivated, by orally administering to asubject a Nanobody or polypeptide of the invention.

Another embodiment of the present invention is a Nanobody or polypeptideof the invention for use in treating, preventing and/or alleviating thesymptoms or disorders susceptible to modulation by a substance thatcontrols platelet mediated aggregation delivered to the vaginal and/orrectal tract.

In a non-limiting example, a formulation according to the inventioncomprises a Nanobody or polypeptide of the invention, in the form of agel, cream, suppository, film, or in the form of a sponge or as avaginal ring that slowly releases the active ingredient over time (suchformulations are described in EP 707473, EP 684814, U.S. Pat. No.5,629,001).

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by asubstance that controls platelet mediated aggregation delivered to thevaginal and/or rectal tract, by vaginally and/or rectally administeringto a subject a Nanobody or polypeptide of the invention.

Another embodiment of the present invention is a use of a Nanobody orpolypeptide of the invention for the preparation of a medicament fortreating, preventing and/or alleviating the symptoms of disorderssusceptible to modulation by a substance that controls platelet mediatedaggregation delivered to the vaginal and/or rectal tract.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the vaginal and/or rectaltract without being said substance being inactivated, by administeringto the vaginal and/or rectal tract of a subject a Nanobody orpolypeptide of the invention.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the bloodstream of a subjectwithout said substance being inactivated, by administering to thevaginal and/or rectal tract of a subject a Nanobody or polypeptide ofthe invention.

Another embodiment of the present invention is a Nanobody or polypeptideof the invention, for use in treating, preventing and/or alleviating thesymptoms of disorders susceptible to modulation by a substance thatcontrols platelet mediated aggregation delivered to the nose, upperrespiratory tract and/or lung.

In a non-limiting example, a formulation according to the invention,comprises a Nanobody or polypeptide of the invention in the form of anasal spray (e.g. an aerosol) or inhaler. Since the Nanobody orpolypeptide of the invention is small, it can reach its target much moreeffectively than therapeutic IgG molecules.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by asubstance that controls platelet mediated aggregation delivered to theupper respiratory tract and lung, by administering to a subject aNanobody or polypeptide of the invention, by inhalation through themouth or nose.

Another embodiment of the present invention is a use of a Nanobody orpolypeptide of the invention for the preparation of a medicament fortreating, preventing and/or alleviating the symptoms of disorderssusceptible to modulation by a substance that controls platelet mediatedaggregation delivered to the nose, upper respiratory tract and/or lung,without said polypeptide being inactivated.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the nose, upper respiratorytract and lung without inactivation, by administering to the nose, upperrespiratory tract and/or lung of a subject a Nanobody or polypeptide ofthe invention.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the bloodstream of a subjectwithout inactivation by administering to the nose, upper respiratorytract and/or lung of a subject a Nanobody or polypeptide of theinvention.

One embodiment of the present invention is a Nanobody or polypeptide ofthe invention for use in treating, preventing and/or alleviating thesymptoms of disorders susceptible to modulation by a substance thatcontrols platelet mediated aggregation delivered to the intestinalmucosa, wherein said disorder increases the permeability of theintestinal mucosa. Because of their small size, a Nanobody orpolypeptide of the invention can pass through the intestinal mucosa andreach the bloodstream more efficiently in subjects suffering fromdisorders which cause an increase in the permeability of the intestinalmucosa.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by asubstance that controls platelet mediated aggregation delivered to theintestinal mucosa, wherein said disorder increases the permeability ofthe intestinal mucosa, by orally administering to a subject a Nanobodyor polypeptide of the invention.

This process can be even further enhanced by an additional aspect of thepresent invention—the use of active transport carriers. In this aspectof the invention, V_(HH) is fused to a carrier that enhances thetransfer through the intestinal wall into the bloodstream. In anon-limiting example, this “carrier” is a second V_(HH) which is fusedto the therapeutic V_(HH). Such fusion constructs are made using methodsknown in the art. The “carrier” V_(HH) binds specifically to a receptoron the intestinal wall which induces an active transfer through thewall.

Another embodiment of the present invention is a use of a Nanobody orpolypeptide of the invention for the preparation of a medicament fortreating, preventing and/or alleviating the symptoms of disorderssusceptible to modulation by a substance that controls platelet mediatedaggregation delivered to the intestinal mucosa, wherein said disorderincreases the permeability of the intestinal mucosa.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the intestinal mucosa withoutbeing inactivated, by administering orally to a subject a Nanobody orpolypeptide of the invention.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the bloodstream of a subjectwithout being inactivated, by administering orally to a subject aNanobody or polypeptide of the invention.

This process can be even further enhanced by an additional aspect of thepresent invention—the use of active transport carriers. In this aspectof the invention, a Nanobody or polypeptide of the invention asdescribed herein is fused to a carrier that enhances the transferthrough the intestinal wall into the bloodstream. In a non-limitingexample, this “carrier” is a V_(HH) which is fused to said polypeptide.Such fusion constructs made using methods known in the art. The“carrier” V_(HH) binds specifically to a receptor on the intestinal wallwhich induces an active transfer through the wall.

One embodiment of the present invention is a Nanobody or polypeptide ofthe invention for use in treating, preventing and/or alleviating thesymptoms of disorders susceptible to modulation by a substance thatcontrols platelet mediated aggregation which is able pass through thetissues beneath the tongue effectively. A formulation of said Nanobodyor polypeptide of the invention, for example, a tablet, spray, drop isplaced under the tongue and adsorbed through the mucus membranes intothe capillary network under the tongue.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by asubstance that controls platelet mediated aggregation which is able passthrough the tissues beneath the tongue effectively, by sublinguallyadministering to a subject a Nanobody or polypeptide of the invention.

Another embodiment of the present invention is a use of a Nanobody orpolypeptide of the invention for the preparation of a medicament fortreating, preventing and/or alleviating the symptoms of disorderssusceptible to modulation by a substance that controls platelet mediatedaggregation which is able to pass through the tissues beneath thetongue.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the tissues beneath the tonguewithout being inactivated, by administering sublingually to a subject aNanobody or polypeptide of the invention.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the bloodstream of a subjectwithout being inactivated, by administering orally to a subject aNanobody or polypeptide of the invention.

One embodiment of the present invention is a Nanobody or polypeptide ofthe invention for use in treating, preventing and/or alleviating thesymptoms of disorders susceptible to modulation by a substance thatcontrols platelet mediated aggregation which is able pass through theskin effectively.

A formulation of said Nanobody or polypeptide of the invention, forexample, a cream, film, spray, drop, patch, is placed on the skin andpasses through.

An aspect of the invention is a method for treating, preventing and/oralleviating the symptoms of disorders susceptible to modulation by asubstance that controls platelet mediated aggregation which is able passthrough the skin effectively, by topically administering to a subject aNanobody or polypeptide of the invention.

Another embodiment of the present invention is a use of a Nanobody orpolypeptide of the invention for the preparation of a medicament fortreating, preventing and/or alleviating the symptoms of disorderssusceptible to modulation by a substance that controls platelet mediatedaggregation which is able pass through the skin effectively.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the skin without beinginactivated, by administering topically to a subject a Nanobody orpolypeptide of the invention.

An aspect of the invention is a method for delivering a substance thatcontrols platelet mediated aggregation to the bloodstream of a subject,by administering topically to a subject a Nanobody or polypeptide of theinvention.

In another embodiment of the present invention, a Nanobody orpolypeptide of the invention further comprises a carrier Nanobody of theinvention (e.g. V_(HH)) which acts as an active transport carrier fortransport of said Nanobody or polypeptide of the invention via the lunglumen to the blood.

A Nanobody or polypeptide of the invention further comprising a carrierthat binds specifically to a receptor present on the mucosal surface(bronchial epithelial cells) resulting in the active transport of thepolypeptide from the lung lumen to the blood. The carrier Nanobody ofthe invention may be fused to the Nanobody or polypeptide of theinvention. Such fusion constructs made using methods known in the artand are describe herein. The “carrier” Nanobody of the invention bindsspecifically to a receptor on the mucosal surface which induces anactive transfer through the surface.

Another aspect of the present invention is a method to determine whichNanobodies of the invention (e.g. V_(HH)s) are actively transported intothe bloodstream upon nasal administration. Similarly, a naïve or immuneV_(HH) phage library can be administered nasally, and after differenttime points after administration, blood or organs can be isolated torescue phages that have been actively transported to the bloodstream. Anon-limiting example of a receptor for active transport from the lunglumen to the bloodstream is the Fc receptor N (FcRn). One aspect of theinvention includes the V_(HH) molecules identified by the method. SuchV_(HH) can then be used as a carrier V_(HH) for the delivery of atherapeutic V_(HH) to the corresponding target in the bloodstream uponnasal administration.

One embodiment of the present invention is a Nanobody or polypeptide ofthe invention for use in treating, preventing and/or alleviating thesymptoms of disorders relating to platelet-mediated aggregation ordysfunction thereof. Said disorders include, thrombotic thrombocytopenicpurpura (TTP), transient cerebral ischemic attack, unstable or stableangina pectoris, cerebral infarction, myocardial infarction, peripheralarterial occlusive disease, restenosis. Said disorders further includethose arising from coronary by-pass graft, coronary artery valvereplacement and coronary interventions such angioplasty, stenting, oratherectomy.

Other disorders are any of the formation of a non-occlusive thrombus,the formation of an occlusive thrombus, arterial thrombus formation,acute coronary occlusion, restenosis, restenosis after PCTA or stenting,thrombus formation in stenosed arteries, hyperplasia after angioplasty,atherectomy or arterial stenting, occlusive syndrome in a vascularsystem or lack of patency of diseased arteries.

One aspect of the invention is a Nanobody or polypeptide of theinvention for use in the treatment, prevention and/or alleviation ofdisorders or conditions relating to platelet-mediated aggregation ordysfunction thereof, wherein said Nanobody or polypeptide of theinvention is administered intravenously, subcutaneously, orally,sublingually, topically, nasally, vaginally, rectally or by inhalation.

Another aspect of the invention is the use of a Nanobody or polypeptideof the invention for the preparation of a medicament for the treatment,prevention and/or alleviation of disorders or conditions relating toplatelet-mediated aggregation or dysfunction thereof, wherein saidNanobody or polypeptide of the invention is administered intravenously,subcutaneously, orally, sublingually, topically, nasally, vaginally,rectally or by inhalation.

Another aspect of the invention is a method of treating, preventingand/or alleviating disorders or conditions relating to relating toplatelet-mediated aggregation or dysfunction thereof, comprisingadministering to a subject a Nanobody or polypeptide of the invention,wherein said heterospecific Nanobody or polypeptide of the invention isadministered intravenously, subcutaneously, orally, sublingually,topically, nasally, vaginally, rectally or by inhalation.

Another aspect of the invention is a Nanobody or polypeptide of theinvention for use in the treatment, prevention and/or alleviation ofdisorders or conditions relating to platelet-mediated aggregation ordysfunction thereof.

Another aspect of the invention is a use of a polypeptide of theinvention for the preparation of a medicament for the treatment,prevention and/or alleviation of disorders or conditions relating toplatelet-mediated aggregation or dysfunction thereof.

One can use a Nanobody or polypeptide of the invention of the presentinvention in order to screen for agents that modulate the binding of thepolypeptide to a vWF. When identified in an assay that measures bindingor said polypeptide displacement alone, agents will have to be subjectedto functional testing to determine whether they act as modulators ofplatelet-mediated aggregation. Some examples of suitable screeningmethods are discussed in WO 04/062551. Of course, these methods caneasily be applied to screening for candidate modulators which alter thebinding between the Nanobody or polypeptide of the invention disclosedherein and vWF.

A cell that is useful according to the invention is preferably selectedfrom the group consisting of bacterial cells such as, for example, E.coli, yeast cells such as, for example, S. cerevisiae, P. pastoris,insect cells or mammalian cells, e.g. as mentioned above.

A cell that is useful according to the invention can be any cell intowhich a nucleic acid sequence encoding a Nanobody or polypeptide of theinvention can be or has been introduced such that the polypeptide isexpressed at natural levels or above natural levels, as defined herein.Preferably a polypeptide of the invention that is expressed in a cellexhibits normal or near normal pharmacology, as defined herein.

According to a preferred embodiment of the present invention, a cell isselected from the group consisting of COS7-cells, a CHO cell, a LM (TK-)cell, a NIH-3T3 cell, HEK-293 cell, K-562 cell or a 1321N1 astrocytomacell but also other transfectable cell lines.

In general, “therapeutically effective amount”, “therapeuticallyeffective dose” and “effective amount” means the amount needed toachieve the desired result or results (treating or preventing plateletaggregation). One of ordinary skill in the art will recognize that thepotency and, therefore, an “effective amount” can vary for the variousNanobodies or polypeptides that inhibit platelet-mediated aggregationused in the invention. One skilled in the art can readily assess thepotency of the Nanobody or polypeptide.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the Nanobody or polypeptidewithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

The invention disclosed herein is useful for treating or preventing acondition of platelet-mediated aggregation, in a subject and comprisingadministering a pharmaceutically effective amount of a Nanobody orpolypeptide or composition that inhibits BTK and that inhibitsplatelet-mediated aggregation.

The invention disclosed herein is useful for treating or preventing thefirst steps of thrombus formation, in a subject and comprisingadministering a pharmaceutically effective amount of a Nanobody orpolypeptide or composition according to the invention.

The invention disclosed herein is useful for treating or preventingrestenosis, in a subject and comprising administering a pharmaceuticallyeffective amount of a Nanobody or polypeptide or composition accordingto the invention.

One aspect of the present invention is the use of Nanobodies orpolypeptides of the invention for treating or preventing a condition ofplatelet-mediated aggregation, in a subject and comprising administeringa pharmaceutically effective amount of a Nanobody or polypeptide incombination with another, such as, for example, aspirin.

One aspect of the present invention is the use of Nanobodies orpolypeptides of the invention for treating or preventing a condition ofplatelet-mediated aggregation, in a subject and comprising administeringa pharmaceutically effective amount of a Nanobody or polypeptide incombination with another, such as, for example, a thrombolytic agent.

Another aspect of the present invention is a use of a Nanobody orpolypeptide of the invention for treating or preventing plaque orthrombus in an individual. Said plaque or thrombus formation may beunder conditions of high sheer. In both thrombosis and reocclusion, thereversible adhesion or tethering of the platelets at high shear rate isfollowed by a firm adhesion through the collagen receptor on plateletsresulting in platelet activation; the tethering of platelets by vWF tocollagen exposed in the damaged vessel wall is especially importantunder high shear conditions. The inventors have found that Nanobody orpolypeptide of the invention of the present invention unexpectedperformed well under high sheer conditions.

The present invention is not limited to the administration offormulations comprising a single Nanobody or polypeptide of theinvention. It is within the scope of the invention to providecombination treatments wherein a formulation is administered to apatient in need thereof that comprises more than one Nanobody orpolypeptide of the invention.

Conditions of platelet-mediated aggregation include, but are not limitedto, unstable angina, stable angina, angina pectoris, embolus formation,deep vain thrombosis, hemolytic uremic syndrome, hemolytic anemia, acuterenal failure, thrombolytic complications, thrombotic thrombocytopenicpurpura, disseminated intravascular comgelopathy, thrombosis, coronaryheart disease, thromboembolic complications, myocardial infarction,restenosis, and atrial thrombosis formation in atrial fibrillation,chronic unstable angina, transient ischemic attacks and strokes,peripheral vascular disease, arterial thrombosis, pre-eclampsia,embolism, restenosis and/or thrombosis following angioplasty, carotidendarterectomy, anastomosis of vascular grafts, and chronic exposure tocardiovascular devices. Such conditions may also result fromthromboembolism and reocclusion during and after thrombolytic therapy,after angioplasty, and after coronary artery bypass.

It is well known in the art how to determine the inhibition ofplatelet-mediated aggregation using the standard tests described herein,or using other similar tests. Preferably, the method would result in atleast a 10% reduction in platelet-mediated aggregation, including, forexample, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or anyamount in between, more preferably by 90%.

Similarly, the method would result in at least a 10% reduction inintracellular calcium mobilisation including, for example, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. Similarly, the methodwould result in at least a 10% reduction in the level of phosphorylatedPLCg 2 including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%.

The reduction can be measured, for example, by comparing the opticalimpedance in a chronology platelet aggregometer. Any other knownmeasurement method may also be used. For example, (1) upon collagenstimulation, the level of collagen-induced intracellular calciummobilization increases over time and so the measurement may includemeasuring the level of collagen-induced intracellular calcium or (2)upon collagen stimulation, the level of phosphorylated PLCg 2 increasesover time and so the measurement may include measuring the level ofphosphorylated PLCg 2.

The cells can be contacted in vitro, for example, by adding a Nanobodyor polypeptide of the invention to the culture medium (by continuousinfusion, by bolus delivery, or by changing the medium to a medium thatcontains the Nanobody or polypeptide) or by adding the Nanobody orpolypeptide to the extracellular fluid in vivo (by local delivery,systemic delivery, inhalation, intravenous injection, bolus delivery, orcontinuous infusion). The duration of “contact” with a cell orpopulation of cells is determined by the time the Nanobody orpolypeptide is present at physiologically effective levels or atpresumed physiologically effective levels in the medium or extracellularfluid bathing the cell or cells. Preferably, the duration of contact is1-96 hours, and more preferably, for 24 hours, but such time would varybased on the half life of the Nanobody or polypeptide and could beoptimized by one skilled in the art using routine experimentation.

The Nanobody or polypeptide useful in the present invention can beformulated as pharmaceutical compositions and administered to amammalian host, such as a human patient or a domestic animal in avariety of forms adapted to the chosen route of administration, i.e.,orally or parenterally, by intra-nasally by inhalation, intravenous,intramuscular, topical or subcutaneous routes.

The Nanobody or polypeptide of the present invention can also beadministered using gene therapy methods of delivery. See, e.g., U.S.Pat. No. 5,399,346, which is incorporated by reference in its entirety.Using a gene therapy method of delivery, primary cells transfected withthe gene for the Nanobody or polypeptide of the present invention canadditionally be transfected with tissue specific promoters to targetspecific organs, tissue, grafts, tumors, or cells.

Thus, the present Nanobody or polypeptide may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, theNanobody or polypeptide may be combined with one or more excipients andused in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 0.1% of theNanobody or polypeptide. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of the Nanobody or polypeptide in such therapeutically usefulcompositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the Nanobody or polypeptide, sucrose or fructose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoringsuch as cherry or orange flavor. Of course, any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, theNanobody or polypeptide may be incorporated into sustained-releasepreparations and devices.

The Nanobody or polypeptide may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the Nanobody orpolypeptide can be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the Nanobodyor polypeptide in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the present Nanobody or polypeptide may beapplied in pure form, i.e., when they are liquids. However, it willgenerally be desirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, hydroxyalkyls or glycols or water-alcohol/glycolblends, in which the present Nanobody or polypeptide can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the Nanobody or polypeptide to the skin are known to the art;for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the Nanobody or polypeptide can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Generally, the concentration of the Nanobody or polypeptide in a liquidcomposition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the Nanobody or polypeptide required for use in treatmentwill vary with the route of administration, the nature of the conditionbeing treated and the age and condition of the patient and will beultimately at the discretion of the attendant physician or clinician.Also the dosage of the Nanobody or polypeptide varies depending on thetarget cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

The invention provides for an agent that is a modulator ofplatelet-mediated aggregation.

The candidate agent may be a synthetic agent, or a mixture of agents, ormay be a natural product (e.g. a plant extract or culture supernatant).A candidate agent according to the invention includes a small moleculethat can be synthesized, a natural extract, peptides, proteins,carbohydrates, lipids etc.

Candidate modulator agents from large libraries of synthetic or naturalagents can be screened. Numerous means are currently used for random anddirected synthesis of saccharide, peptide, and nucleic acid basedagents. Synthetic agent libraries are commercially available from anumber of companies including Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich (Milwaukee, Wis.). Combinatoriallibraries are available and can be prepared. Alternatively, libraries ofnatural agents in the form of bacterial, fungal, plant and animalextracts are available from e.g., Pan Laboratories (Bothell, Wash.) orMycoSearch (NC), or are readily producible by methods well known in theart. Additionally, natural and synthetically produced libraries andagents are readily modified through conventional chemical, physical, andbiochemical means.

Useful agents may be found within numerous chemical classes. Usefulagents may be organic agents, or small organic agents. Small organicagents have a molecular weight of more than 50 yet less than about 2,500daltons, preferably less than about 750, more preferably less than about350 daltons. Exemplary classes include heterocycles, peptides,saccharides, steroids, and the like. The agents may be modified toenhance efficacy, stability, pharmaceutical compatibility, and the like.Structural identification of an agent may be used to identify, generate,or screen additional agents. For example, where peptide agents areidentified, they may be modified in a variety of ways to enhance theirstability, such as using an unnatural amino acid, such as a D-aminoacid, particularly D-alanine, by functionalizing the amino or carboxylicterminus, e.g. for the amino group, acylation or alkylation, and for thecarboxyl group, esterification or amidification, or the like.

For primary screening, a useful concentration of a candidate agentaccording to the invention is from about 10 mM to about 100 μM or more(i.e. 1 mM, 10 mM, 100 mM, 1 M etc.). The primary screeningconcentration will be used as an upper limit, along with nine additionalconcentrations, wherein the additional concentrations are determined byreducing the primary screening concentration at half-log intervals (e.g.for 9 more concentrations) for secondary screens or for generatingconcentration curves.

A high throughput screening kit according to the invention comprises allthe necessary means and media for performing the detection of an agentthat modulates platelet-mediated aggregation by interacting with atarget of the invention, such as for example vWF, or fragment thereof inthe presence of a polypeptide, preferably at a concentration in therange of 1 μM to 1 mM. The kit comprises the following. Recombinantcells of the invention, comprising and expressing the nucleotidesequence encoding vWF, or fragment thereof, which are grown according tothe kit on a solid support, such as a microtiter plate, more preferablya 96 well microtiter plate, according to methods well known to theperson skilled in the art especially as described in WO 00/02045.Alternatively vWF, or fragment thereof is supplied in a purified form tobe immobilized on, for example, a 96 well microtiter plate by the personskilled in the art. Alternatively vWF, or fragment thereof is suppliedin the kit pre-immobilized on, for example, a 96 well microtiter plate.Modulator agents according to the invention, at concentrations fromabout 1 μM to 1 mM or more, are added to defined wells in the presenceof an appropriate concentration of Nanobody or polypeptide of theinvention said concentration of said polypeptide preferably in the rangeof 1 μM to 1 mM. Kits may contain more than one polypeptide

Binding assays are performed as according to the methods alreadydisclosed herein and the results are compared to the baseline level of,for example vWF, or fragment thereof binding to a polypeptide of theinvention, but in the absence of added modulator agent. Wells showing atleast 2 fold, preferably 5 fold, more preferably 10 fold and mostpreferably a 100 fold or more increase or decrease in vWF-polypeptidebinding (for example) as compared to the level of activity in theabsence of modulator, are selected for further analysis.

The invention provides for kits useful for screening for modulators ofplatelet-mediated aggregation, as well as kits useful for diagnosis ofdiseases or disorders characterised by dysregulation platelet-mediatedaggregation. Kits useful according to the invention can include anisolated vWF, or fragment thereof. Alternatively, or in addition, a kitcan comprise cells transformed to express vWF, or fragment thereof. In afurther embodiment, a kit according to the invention can comprise apolynucleotide encoding vWF, or fragment thereof. In a still furtherembodiment, a kit according to the invention may comprise the specificprimers useful for amplification of vWF, or fragment thereof. Kitsuseful according to the invention can comprise a Nanobody or polypeptideof the invention. A kit according to the invention can comprise cellstransformed to express said polypeptide. Kits may contain more than onepolypeptide. In a further embodiment, a kit according to the inventioncan comprise a polynucleotide encoding a macromolecule, for example,vWF, or fragment thereof. In a still further embodiment, a kit accordingto the invention may comprise the specific primers useful foramplification of a macromolecule such as, for example, vWF, or fragmentthereof. All kits according to the invention will comprise the stateditems or combinations of items and packaging materials therefore. Kitswill also include instructions for use.

The invention also provides for invasive medical devices coated with aNanobody or polypeptide of the invention or an agent resulting from ascreening method of the invention for use in devices requiring the same.Non-limiting examples of devices include surgical tubing, occlusiondevices, prosthetic devices. Application for said devices includesurgical procedures which require a modulation of platelet-mediatedaggregation around the site of invasion.

One embodiment of the present is a method for treating invasive medicaldevices to prevent platelet-mediate aggregation around the site ofinvasion comprising the step of coating said device with a Nanobody orpolypeptide of the invention or agent according to the invention.

Another embodiment of the present is a invasive medical devices thatcircumvents platelet-mediate aggregation around the site of invasion,wherein said device is coated with a Nanobody or polypeptide of theinvention or agent according to the invention.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one aggregation-mediated disorder (asdescribed herein), said method comprising administering, to a subject inneed thereof, a pharmaceutically active amount of a Nanobody of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk from, the diseases anddisorders mentioned herein.

The invention also relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering a Nanobody or polypeptide of theinvention to a patient, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of a Nanobodyof the invention, of a polypeptide of the invention, and/or of apharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of a Nanobody of the invention, of apolypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In another embodiment, the invention relates to a method forimmunotherapy, and in particular for passive immunotherapy, which methodcomprises administering, to a subject suffering from or at risk of thediseases and disorders mentioned herein, a pharmaceutically activeamount of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the above methods, the Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same can beadministered in any suitable manner, depending on the specificpharmaceutical formulation or composition to be used. Thus, theNanobodies and/or polypeptides of the invention and/or the compositionscomprising the same can for example be administered orally,intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly,or via any other route of administration that circumvents thegastrointestinal tract), intranasally, transdermally, topically, bymeans of a suppository, by inhalation, again depending on the specificpharmaceutical formulation or composition to be used. The clinician willbe able to select a suitable route of administration and a suitablepharmaceutical formulation or composition to be used in suchadministration, depending on the disease or disorder to be prevented ortreated and other factors well known to the clinician.

As mentioned herein and as will be clear to the skilled person, foracute conditions and complications (i.e. as may occur with some of theaggregation-mediated disorders mentioned herein), usually administrationdirectly into the blood stream such by infusion or injection or anyother suitable means will be preferred.

The Nanobodies and/or polypeptides of the invention and/or thecompositions comprising the same are administered according to a regimeof treatment that is suitable for preventing and/or treating the diseaseor disorder to be prevented or treated. The clinician will generally beable to determine a suitable treatment regimen, depending on factorssuch as the disease or disorder to be prevented or treated, the severityof the disease to be treated and/or the severity of the symptomsthereof, the specific Nanobody or polypeptide of the invention to beused, the specific route of administration and pharmaceuticalformulation or composition to be used, the age, gender, weight, diet,general condition of the patient, and similar factors well known to theclinician.

Generally, the treatment regimen will comprise the administration of oneor more Nanobodies and/or polypeptides of the invention, or of one ormore compositions comprising the same, in one or more pharmaceuticallyeffective amounts or doses. The specific amount(s) or doses toadministered can be determined by the clinician, again based on thefactors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific Nanobody andpolypeptide of the invention to be used, the specific route ofadministration and the specific pharmaceutical formulation orcomposition used, the Nanobodies and polypeptides of the invention willgenerally be administered in an amount between 1 gram and 0.01 microgramper kg body weight per day, preferably between 0.1 gram and 0.1microgram per kg body weight per day, such as about 1, 10, 100 or 1000microgram per kg body weight per day, either continuously (e.g. byinfusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, for example on the basis of the factors cited above andhis expert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, half-life and similar factors well known to the skilledperson.

Usually, in the above method, a single Nanobody or polypeptide of theinvention will be used. It is however within the scope of the inventionto use two or more Nanobodies and/or polypeptides of the invention incombination.

The Nanobodies and polypeptides of the invention may also be used incombination with one or more further pharmaceutically active compoundsor principles, i.e. as a combined treatment regimen, which may or maynot lead to a synergistic effect. Again, the clinician will be able toselect such further compounds or principles, as well as a suitablecombined treatment regimen, based on the factors cited above and hisexpert judgement.

In particular, the Nanobodies and polypeptides of the invention may beused in combination with other pharmaceutically active compounds orprinciples that are or can be used for the prevention and/or treatmentof the diseases and disorders cited herein, as a result of which asynergistic effect may or may not be obtained. Examples of suchcompounds and principles, as well as routes, methods and pharmaceuticalformulations or compositions for administering them will be clear to theclinician, and for example include, but are not limited to heparin,aspirin (e.g. Aspegic®), Plavix and/or Reopro.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are administered to be simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and or a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk from, the diseases anddisorders mentioned herein.

The invention also relates to the use of a Nanobody or polypeptide ofthe invention in the preparation of a pharmaceutical composition for theprevention and/or treatment of at least one disease or disorder (e.g. anaggregation disorder as mentioned herein) that can be prevented and/ortreated by administering a Nanobody or polypeptide of the invention to apatient.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

Finally, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto other “scaffolds”, including but notlimited to human scaffolds or non-immunoglobulin scaffolds. Suitablescaffolds and techniques for such CDR grafting will be clear to theskilled person and are well known in the art, see for example U.S. Pat.No. 7,180,370, WO 01/27160, EP 0 605 522, EP 0 460 167, U.S. Pat. No.7,054,297, Nicaise et al., Protein Science (2004), 13:1882-1891; Ewertet al., Methods, 2004 October; 34(2):184-199; Kettleborough et al.,Protein Eng. 1991 October; 4(7): 773-783; O'Brien and Jones, MethodsMol. Biol. 2003:207:81-100; and Skerra, J. Mol. Recognit.2000:13:167-187, and Saerens et al., J. Mol. Biol. 2005 Sep. 23;352(3):597-607, and the further references cited therein; and alsoinclude for example the framework regions of other (single) domainantibodies. For example, techniques known per se for grafting mouse orrat CDR's onto human frameworks and scaffolds can be used in ananalogous manner to provide chimeric proteins comprising one or more ofthe CDR's of the Nanobodies of the invention and one or human frameworkregions or sequences.

Thus, in another embodiment, the invention comprises a chimericpolypeptide comprising at least one CDR sequence chosen from the groupconsisting of CDR1 sequences, CDR2 sequences and CDR3 sequencesmentioned herein for the Nanobodies of the invention. Preferably, such achimeric polypeptide comprises at least one CDR sequence chosen from thegroup consisting of the CDR3 sequences mentioned herein for theNanobodies of the invention, and optionally also at least one CDRsequence chosen from the group consisting of the CDR1 sequences and CDR2sequences mentioned herein for the Nanobodies of the invention. Forexample, such a chimeric polypeptide may comprise one CDR sequencechosen from the group consisting of the CDR3 sequences mentioned hereinfor the Nanobodies of the invention, one CDR sequence chosen from thegroup consisting of the CDR1 sequences mentioned herein for theNanobodies of the invention and one CDR sequence chosen from the groupconsisting of the CDR1 sequences and CDR2 sequences mentioned herein forthe Nanobodies of the invention. The combinations of CDR's that arementioned herein as being preferred for the Nanobodies of the inventionwill usually also be preferred for these chimeric polypeptides.

In said chimeric polypeptides, the CDR's may be linked to further aminoacid sequences and/or may be linked to each other via amino acidsequences, in which said amino acid sequences are preferably frameworksequences or are amino acid sequences that act as framework sequences,or together form a scaffold for presenting the CDR's. Reference is againmade to the prior art mentioned in the last paragraph. According to onepreferred embodiment, the amino acid sequences are human frameworksequences, for example V_(H)3 framework sequences. However, non-human,synthetic, semi-synthetic or non-immunoglobulin framework sequences mayalso be used. Preferably, the framework sequences used are such that (1)the chimeric polypeptide is capable of binding xxxx, i.e. with anaffinity that is at least 1%, preferably at least 5%, more preferably atleast 10%, such as at least 25% and up to 50% or 90% or more of theaffinity of the corresponding Nanobody of the invention; (2) thechimeric polypeptide is suitable for pharmaceutical use; and (3) thechimeric polypeptide is preferably essentially non-immunogenic under theintended conditions for pharmaceutical use (i.e. indication, mode ofadministration, dosis and treatment regimen) thereof (which may beessentially analogous to the conditions described herein for the use ofthe Nanobodies of the invention).

According to one non-limiting embodiment, the chimeric polypeptidecomprises at least two CDR sequences (as mentioned above) linked via atleast one framework sequence, in which preferably at least one of thetwo CDR sequences is a CDR3 sequence, with the other CDR sequence beinga CDR1 or CDR2 sequence. According to a preferred, but non-limitingembodiment, the chimeric polypeptide comprises at least two CDRsequences (as mentioned above) linked at least two framework sequences,in which preferably at least one of the three CDR sequences is a CDR3sequence, with the other two CDR sequences being CDR1 or CDR2 sequences,and preferably being one CDR1 sequence and one CDR2 sequence. Accordingto one specifically preferred, but non-limiting embodiment, the chimericpolypeptides have the structure FR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4′, inwhich CDR1, CDR2 and CDR3 are as defined herein for the CDR's of theNanobodies of the invention, and FR1′, FR2′, FR3′ and FR4′ are frameworksequences. FR1′, FR2′, FR3′ and FR4′ may in particular be Framework 1,Framework 2, Framework 3 and Framework 4 sequences, respectively, of ahuman antibody (such as V_(H)3 sequences) and/or parts or fragments ofsuch Framework sequences. It is also possible to use parts or fragmentsof a chimeric polypeptide with the structureFR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4. Preferably, such parts or fragmentsare such that they meet the criteria set out in the preceding paragraph.

The invention will now be further described by means of the followingnon-limiting examples and figures, in which the Figures show:

FIG. 1: Binding of nanobodies to vWF in ELISA

FIG. 2: Alignment of 12A5 homologue nanobody sequences (12A5, SEQ IDNO:60; 12B4, SEQ ID NO:67; 12E8, SEQ ID NO:68; 12A6, SEQ ID NO:69; 12D8,SEQ ID NO:70)

FIG. 3: Alignment of 12B6 homologue nanobody sequences (12B6, SEQ IDNO:62; 12A2, SEQ ID NO:71; 12F2, SEQ ID NO:72; 14H10, SEQ ID NO:73)

FIG. 4: Binding of 12A5 homologue nanobodies to vWF in BIACORE

FIG. 5: Binding of 12B6 homologue nanobodies to vWF in BIACORE

FIG. 6: Platelet adhesion at different concentrations of 12B6, 12A2 and12A5 nanobodies

FIG. 7 a: Binding in ELISA to vWF for 12B6 nanobody after heating atincreasing temperatures

FIG. 7 b: Binding in ELISA to vWF for 12A2 nanobody after heating atincreasing temperatures

FIG. 7 c: Binding in ELISA to vWF for 12A5 nanobody after heating atincreasing temperatures

FIG. 8 a: Binding of vWF from different species to 12B6 nanobody inELISA

FIG. 8 b: Binding of vWF from different species to 12A2 nanobody inELISA

FIG. 8 c: Binding of vWF from different species to 12A5 nanobody inELISA

FIG. 9: Binding of bivalent 12B6 nanobodies to vWF in BIACORE

FIG. 10: Binding of bivalent 12A2 nanobodies to vWF in BIACORE

FIG. 11: Binding of bivalent 12A5 nanobodies to vWF in BIACORE

FIG. 12: Binding in ELISA to vWF of bivalent 12B6 nanobodies afterheating at increasing temperatures

FIG. 13: Binding in ELISA to vWF of bivalent 12A2 nanobodies afterheating at increasing temperatures

FIG. 14: Binding in ELISA to vWF of bivalent 12A5 nanobodies afterheating at increasing temperatures

FIG. 15: Alignment of humanised 12B6 nanobody sequences (12B6, SEQ IDNO:62; 12B6H1, SEQ ID NO:86; 12B6H2, SEQ ID NO:87; 12B6H3, SEQ ID NO:88;12B6H4, SEQ ID NO:89)

FIG. 16: Binding in ELISA to vWF of wild type and humanised 12B6nanobody

FIG. 17: Alignment of humanised 12A2 nanobody sequences (12A2, SEQ IDNO:71, 12A2H1, SEQ ID NO:90, 12A2H3, SEQ ID NO:91; 12A2H4, SEQ ID NO:92;12A2H11, SEQ ID NO:93; 12A2H13, SEQ ID NO:94)

FIG. 18: Binding in ELISA to vWF of humanised 12A2 nanobodies, afterheating at increasing temperatures

FIG. 19: Binding in ELISA to vWF of humanised 12A2 nanobodies

FIG. 20: Alignment of humanised 12A5 nanobody sequences (12A5, SEQ IDNO:60; 12A5H1, SEQ ID NO:95; 12A5H2, SEQ ID NO:96; 12A5H3, SEQ ID NO:97)

FIG. 21: Binding in ELISA to vWF of wild type and humanised 12A5nanobody

FIG. 22: Alignment of nanobodies selected for bivalent form (12A2H1, SEQID NO:90; 12A2H4, SEQ ID NO:92; 12B6H2, SEQ ID NO:87)

FIG. 23: Platelet adhesion at different concentrations of bivalent(humanised) nanobodies

FIG. 24: Blood flow pattern for Folts model in baboons

FIG. 25: Experimental setup for Folts model in baboons

FIG. 26: Folts study of baboon control group. The blood flow in functionof time is shown, indicating the CFRs (representative of 2 independentexperiments)

FIG. 27: Folts study of baboon group treated with Aspegic. The bloodflow in function of time is shown, indicating the CFRs (representativeof 3 independent experiments)

FIG. 28: study of baboon group treated with Heparin. The blood flow infunction of time is shown, indicating the CFRs (representative of 3independent experiments)

FIG. 29: Folts study of baboon group treated with Plavix. The blood flowin function of time is shown, indicating the CFRs (representative of 4independent experiments)

FIG. 30: Folts study of baboon group treated with Reopro. The blood flowin function of time is shown, indicating the CFRs (representative of 3independent experiments)

FIG. 31: Folts study of baboon group treated with ALX-0081 (SEQ IDNO:98). The blood flow in function of time is shown, indicating the CFRs(representative of 8 independent experiments)

FIG. 32: Flow read out from baboon ID 6 treated with a combination ofAspegic, Heparin, Plavix and ALX-0081

FIG. 33: Averages of relative blood loss in function of different dosesof Plavix, Reopro and ALX-0081

FIG. 34: Average length of CFRs and average relative amount of bloodloss for animals treated with Plavix in function of increasing drugdose.

FIG. 35: Average length of CFRs and average relative amount of bloodloss for animals treated with Reopro in function of increasing drug dose

FIG. 36: Average length of CFRs and average relative amount of bloodloss for animals treated with ALX-0081 in function of increasing drugdose

FIG. 37: ristocetin-induced aggregation (%, ▪) and length of CFRs (s,♦)for each baboon treated with ALX-0081 in function of all doses

FIG. 38: Concentration of ALX-0081 in plasma versus the length of CFRsfor all baboons treated with ALX-0081

FIG. 39: Concentration of ALX-0081 in plasma versus relative amount ofblood loss from the gauzes

FIG. 40: Folts study of baboon 1 treated with ALX-0081 and vWF. Theblood flow in function of time is shown, indicating the CFRs

FIG. 41: strings (arrows) of adhered platelets on ULvWF secreted fromstimulated endothelial cells

FIG. 42: Absence of strings when platelets are perfused over ULvWF inthe presence of ALX-0081

FIG. 43: Control perfusion experiment: ULvWF strings before (panel A,indicated with red arrows) and during (panel B) perfusion with normalplasma. In panel B, ULvWF strings being cleaved by ADAMTS-13 areindicated with a blue and red arrow for a piece of an ULvWF stringmoving away or for largely cleaved ULvWF strings respectively

FIG. 44: Perfusion experiment in presence of ALX-0081. Microscopic imageof a field before (panel A) and of the same field after (panel B)perfusion with normal plasma. An ULvWF string is indicated in panel Awith a red arrow which is absent in panel B due to cleavage of the ULvWFby ADAMTS-13.

FIG. 45: cleavage of A1-A2-A3 by ADAMTS-13 present in normal pool plasma(NPP) in the absence and presence of ALX-0081

and in which the Tables, which form an integral part of the presentdescription, are as follows:

Table 8: Sequence listing of anti-vWF nanobodies

Table 9: Expression yields of anti-vWF nanobodies

Table 10: Platelet adhesion in perfusion chamber of anti-vWF nanobodies

Table 11: Sequence listing of 12B6 and 12A5 homologue nanobodies

Table 12: Estimated K-on, K-off and KD values for 12A5 homologuenanobodies

Table 13: Estimated K-on, K-off and KD values for 12B6 homologuenanobodies

Table 14: Real KD value of 12B6, 12A2 and 12A5 nanobodies

Table 15: Platelet adhesion in perfusion chamber of 12B6, 12A2 and 12A5nanobodies

Table 16: Concentration of 12B6, 12A2 and 12A5 nanobodies after heatingat increasing temperatures

Table 17: Sequence listing of bivalent nanobodies

Table 18: Sequence listing of linker sequences

Table 19: Expression yields of bivalent 12B6, 12A2 and 12A5 nanobodies

Table 20: Concentration of 12B6 bivalent nanobodies after heating atincreasing temperatures

Table 21: Concentration of 12A2 bivalent nanobodies after heating atincreasing temperatures

Table 22: Concentration of 12A5 bivalent nanobodies after heating atincreasing temperatures

Table 23: Platelet adhesion in perfusion chamber of 12A2 bivalentnanobodies

Table 24: Sequence listing of humanised 12B6 nanobodies

Table 25: Expression yields of wild type and humanised 12B6 nanobodies

Table 26: Concentration of wild type and humanised 12B6 nanobodies afterheating at increasing temperatures

Table 27: KD values for wild type and humanised 12B6 nanobodies

Table 28: Sequence listing of humanised 12A2 nanobodies

Table 29: Expression yields of wild type and humanised 12A2 nanobodies

Table 30: Concentration of wild type and humanised 12A2 nanobodies afterheating at increasing temperatures

Table 31: Platelet adhesion of wild type and humanised 12A2 nanobodiesin perfusion chamber at 0.7 and 1.5 ug/ml

Table 32: Platelet adhesion of wild type and humanised 12A2 nanobodiesin perfusion chamber at 0.5, 1 and 2 ug/ml

Table 33: KD values for wild type and humanised 12A2 nanobodies

Table 34: Sequence listing of humanised 12A5 nanobodies

Table 35: Expression yields of wild type and humanised 12A5 nanobodies

Table 36: Concentration of wild type and humanised 12A5 nanobodies afterheating at increasing temperatures

Table 37: KD values for wild type and humanised 12A5 nanobodies

Table 38: Sequence listing of humanised bivalent nanobodies Table 39:Expression yields of humanised bivalent nanobodies

Table 40: Concentration of humanised bivalent nanobody after heating atincreasing temperatures

Table 41: Platelet adhesion of wild type and humanised bivalentnanobodies

Table 42: baboons used with the different test compounds in the Foltsstudy

Table 43: Length of CFRs (s) for control animals (ND=not done)

Table 44: Length of CFRs (s) for animals treated with Aspegic™ (ND=notdone)

Table 45: Length of CFRs (s) for animals treated with Heparin™ (ND=notdone)

Table 46: Length of CFRs (s) for animals treated with Plavix™ (ND=notdone)

Table 47: Length of CFRs (s) for animals treated with Reopro™ (ND=notdone)

Table 48: Length of CFRs (s) for animals treated with ALX-0081 (ND=notdone)

Table 49: baboons used with the different test compounds in the Foltsstudy

Table 50: Inhibition of CFRs in the Folts model for the different drugstested. The number of experiments in which an inhibition of CFRs wasobserved in the mentioned different conditions is shown as a function ofthe total number of independent repeats of that condition.Table 51: Length of CFRs (seconds) for each baboon and each dose ofAspegic, Heparin, Plavix and ALX-0081. The effective dose is indicatedin yellowTable 52: Blood loss relative to the second control gauze for animalstreated with Plavix™ in function of final dose (STD=standard deviation)Table 53: Blood loss relative to the second control gauze for animalstreated with Reopro™ in function of final dose (STD=standard deviation)Table 54: Blood loss relative to the second control gauze for animalstreated with ALX-0081 in function of final dose (STD=standard deviation)Table 55: The average of the total amount of blood loss (=sum of bloodloss from the first five doses of test compound) as relative to thesecond control gauzeTable 56: Blood loss in gauzes relative to the second control gauze foreach baboon treated with Aspegic, Heparin, Plavix and ALX-0081 infunction of drug dose. The effective drug dose in which a completeinhibition of CFRs was observed, is indicated in yellowTable 57: % ristocetin-induced platelet aggregation for each baboontreated with Aspegic, Heparin, Plavix and ALX-0081 in function of drugdoseTable 58: concentration of ALX-0081 [μg/ml] in blood samples obtained at10 minutes after administrationTable 59: Length of CFRs [seconds] for baboons treated with ALX-0081 andwith vWFTable 60: Volumes [α] to prepare the different mixtures for study ofcleavage of A1A2A3 by ADAMTS13.

EXAMPLES A. Selection and Screening of Nanobodies Specific for vWF andInhibiting Platelet Adhesion Example 1 Antigen Specific MonovalentNanobodies

The nanobodies represented in Table 8 SEQ ID Nos: 60 to 66 are obtainedfrom llamas immunized with human vWF or with recombinant A1 domain ofvWF. The nanobodies bind to the A1 domain of vWF and inhibit theinteraction between vWF and gpIb on the platelets.

Example 2 Expression and Purification of Nanobodies

Plasmid was prepared (QIAGEN, according to the manufacturersinstructions) and was transformed into WK6 or TG1 electro-competentcells. A single colony was used to start an overnight culture in LBcontaining 2% glucose and 100 μg/ml ampicillin. This overnight culturewas diluted 100-fold in 2×300 ml TB medium containing 100 μg/mlampicillin, and incubated at 37° C. until OD600 nm=0.5. 1 mM IPTG wasadded and the culture was incubated for 3 more hours at 37° C. orovernight at 28° C.

Cultures were centrifuged for 20 minutes at 10000 rpm at 4° C. Thepellet was frozen overnight or for 1 hour at −20° C. Next, the pelletwas thawed at room temperature for 40 minutes, resuspended in 20 ml peribuffer (50 mM NaH₂PO₄ and 300 mM NaCl) and shaken at room temperaturefor 1 hour. Periplasmic fraction was isolated by centrifugation for 20minutes at 4° C. at 20000 rpm. The nanobodies were purified on a Nickelcolumn (TALON, Clonetech) as described by the manufacturer andexpression yields were calculated as represented in Table 9.

Example 3 Binding of nanobodies to vWF in ELISA

The nanobodies of Example 1 were tested for binding to vWF in ELISA.Therefore, a microtiterplate (Nunc, Maxisorb) was coated with vWF (RedCross) at a 200-fold dilution and pre-warmed for 15 minutes at 37° C.The plate was coated overnight at 4° C. The plate was then washed withPBS-Tween and blocked for two hours at room temperature with PBS-1%casein. After washing, the samples were applied starting at aconcentration of 10 μg/ml and 3-fold dilutions were made in PBS. After atwo hours incubation period, the plates were washed and mouse monoclonalanti-myc antibody at a 1000-fold dilution was applied for 1 hour at roomtemperature. The plates were washed and polyclonal anti-mouse-HRP (DAKO)was applied at a 1000-fold dilution for one hour at room temperature.The plates were washed and ABTS/H₂O₂ substrate was applied. The OD 405nm was measured. Results are shown in FIG. 1.

Example 4 Inhibition of Platelet Adhesion by Nanobodies in a FlowChamber

The protein samples were analysed in a perfusion chamber. Thermanoxcoverslips (Nunc) were soaked overnight in 80% ethanol, rinsedthoroughly with distilled water and air-dried. Human placental collagentype III (Sigma) was solubilized in 0.05 mol/l acetic acid and sprayedon the coverslips at a final density of 30 μg/cm² with a retouchingairbrush. After spraying the coverslips were blocked with 1% humanalbumin solution in PBS for at least 1 hour at RT. Perfusions wereperformed with a single-pass perfusion chamber under non-pulsatile flowconditions using a modified small perfusion chamber with a slit heightof 0.1 mm and a slit width of 2 mm. Blood was obtained by venipuncturefrom healthy volunteers and anti-coagulated with Penta/PPACK. Triplicatecoverslips were inserted into the chamber. Five milliliters of blood waspre-warmed at 37° C. for 5 minutes with or without addition of 2microgram/ml nanobody and then circulated through the chamber for 5minutes at a wall shear rate of 1600 s⁻¹ using an infusion pump. After aperfusion run, the coverslip was taken from the chamber, rinsed in Hepesbuffered saline (10 mM Hepes, 150 mM NaCL, ph 7.4), fixed in 0.5%glutaraldehyde in PBS, dehydrated in methanol and stained withMay-Grünwald and Giemsa (Riedel de Haën). Platelet deposition wasevaluated as platelet surface coverage using light microscopy andcomputer-assisted analysis. Results are shown in Table 10. Nanobodies12B6 and 12A5 clearly inhibit platelet adhesion to collagen type III inthe perfusion chamber at high shear rate.

Example 5 Analysis in BIACORE for Binding to vWF for HomologuesNanobodies

Nanobodies 12B6 and 12A5 inhibit platelet adhesion in the perfusionchamber. Homologue sequences were obtained from the llama comprising theamino acid differences as shown in Table 11 SEQ ID Nos 67 to 73. FIGS. 2and 3 represent the alignment of the 12A5 and 12B6 homologue nanobodysequences.

vWF was covalently bound to the sensor chip surface via amine coupling.The CM5 surface of the chip was activated by the injection of EDC/NHS(1:1 mix of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.1M N-hydroxysuccinimide in water) for 7 minutes. Upon activation vWF wasinjected until an increase of 6000 response units was detected. Theexcess of reactive groups was deactivated with 1 M Ethanolamine-HCl (pH8.5) for 7 minutes. The flowrate was kept constant during theimmobilization procedure at 5 ul/min. The eluent buffer was 0.01 M HEPES(pH 7,4) with 0.15 M NaCl, 3 mM EDTA and 0.005% Surfactant P20.

The nanobodies 12B6 and 12A5 and their homologue proteins were analysedin BIACORE on vWF at a concentration of the nanobody of 5 μg/ml as shownin FIGS. 4 and 5. Estimated kon, K-off and KD values are represented inTable 12 and 13. Nanobodies 12A5 and 12B5 have the best K-on and K-offrates. Nanobodies 12A2 and 12B6 have the best K-on rate, the K-off ratesare very comparable for all nanobodies tested.

Table 14 shows the real KD value for vWF on BIACORE using a range ofconcentrations of the nanobodies. From the set of curves that weregenerated for each nanobody, only those curves where equilibrium wasreached, were used to derive the KD value via steady state affinity.

For the treatment of acute events, the fast inhibition of vWF is veryimportant, and thus a fast K-on rate is preferred. The K-on ratedetermines how fast a nanobody binds its target (vWF) when injected intohuman or animals.

Example 6 Compare Inhibiting Nanobodies for Potency in the PerfusionChamber

To compare the potency for inhibition of platelet adhesion, thenanobodies 12A2, 12B6 and 12A5 were tested in the perfusion chamber at0.2, 0.4 and 0.6 μg/ml. The experiment was performed using the samedonor for all nanobodies. Results are shown in Table 15 and FIG. 6. Thenanobodies show a very comparable inhibiting capacity in the perfusionchamber, with full inhibition of platelet adhesion at a concentration of0.6 μg/ml nanobody.

Example 7 Stability of Nanobodies at Elevated Temperatures

A stock solution of nanobodies at a concentration of 200 μg/ml in PBSwas prepared and divided into several tubes. Each tube containingnanobody was incubated at different temperatures for 1 hour, then cooledat room temperature for 2 hours and put at 4° C. overnight. The nextday, the samples were centrifuged for 30 minutes at 13000 rpm, and thesupernatant was tested for OD280 nm. The concentration of supernatantswere measured spectrophotometrically and expressed as a percentage ofthe concentration at room temperature. The results are summarized inTable 16.

The supernatants were also tested in ELISA for binding to vWF asdescribed above in Example 3. As shown in FIG. 7 a (12B6), 7 b (12A2)and 7 c (12A5), the nanobodies are very stable at elevated temperatures.

Example 8 Cross-Reactivity of the Nanobodies with vWF from Other Species

A microtiterplate was coated with mouse anti-myc at 1/1000 overnight at4° C. The plate was washed with PBS-Tween and blocked for two hours atroom temperature with PBS-1% casein. After washing, the nanobodies wereapplied at a concentration of 10 μg/ml in PBS. After a one hourincubation period, the plates were washed and plasma (dog, pig, human,baboon and cynomologues monkey) was applied starting at a five-folddilution and making further two-fold dilutions in PBS. The plates wereincubated for 1 hour at room temperature. The plates were washed andpolyclonal anti-vWF-HRP (DAKO) was applied at a 2000-fold dilution forone hour at room temperature. The plates were washed and ABTS/H₂O₂substrate was applied. The OD 405 nm was measured.

As shown in FIG. 8 a (12B6), 8 b (12A2) and 8 c (12A5), nanobodies 12A5,12A2 and 12B6 are cross-reactive with human, baboon and cynomologuesmonkey vWF. The nanobodies 12A2 and 12B6 are also cross-reactive withpig vWF. These nanobodies can therefore be tested for efficacy andsafety in pigs. None of the nanobodies is cross-reactive with dog vWF.

B. Construction of Bivalent Nanobodies Specific for vWF and InhibitingPlatelet Adhesion Example 9 Amino Acid Sequences of the BivalentNanobodies

Table 17 SEQ ID Nos 74 to 82 represents bivalent nanobodies constructedfor 12B6, 12A2 and 12A5. The nanobodies were linked with the linkersrepresented in Table 18 SEQ ID Nos 83 to 85.

Example 10 Expression and Purification of the Bivalent Nanobodies

Expressions were performed as described above in Example 2. Expressionyields are summarized in Table 19.

Example 11 Analysis of the Bivalent Nanobodies in BIACORE

The bivalent nanobodies of Example 9 were analyzed in BIACORE at 1.3 nMas described above in Example 5 to compare the affinities for vWF versusthe monovalent nanobody. The bivalent nanobodies 12B6 (FIG. 9), 12A2(FIG. 10) and 12A5 (FIG. 11) have an improved affinity for vWF whencompared to the monovalent nanobody.

Example 12 Stability of the Bivalent Nanobodies at Elevated Temperatures

Stability of the bivalent nanobodies was measured as described above inExample 7. The concentration (μg/ml) of the supernatants was measuredand expressed as a percentage of the concentration at room temperature.The results are summarized in Table 20 (bivalent 12B6), Table 21(bivalent 12A2) and Table 22 (bivalent 12A5).

The supernatants were tested in ELISA for binding to vWF as describedabove in Example 3. The supernatants were applied starting at a 1/100dilution and 1/5 dilutions were made in PBS. Results are shown in FIG.12 (12B6), FIG. 13 (12A2) and FIG. 14 (12A5).

Example 13 Analysis of Monovalent and Bivalent 12A2 in the Flow Chamber

Nanobody 12A2 (monovalent and bivalent forms) was tested in theperfusion chamber as described above in Example 4. The experiment wasperformed using the same donor for all nanobodies. Results aresummarized in Table 23. The bivalent nanobodies inhibit plateletadhesion more efficiently then the monovalent form.

C. Humanisation of Nanobodies Specific for vWF and Inhibiting PlateletAdhesion Example 14 Humanisation of 12B6 Nanobody

Table 24 SEQ ID Nos: 86 to 89 represents four humanised 12B6 nanobodies.Table II lists the amino acid changes that were performed to achievethese sequences. FIG. 15 represents the alignment of the humanisedsequences for 12B6.

TABLE II non-limiting humanizing substitutions Q1E A14P A17S E44G D46ER76N M77S A82bS K83R P84A 12B6H1 X X X X X X X 12B6H2 X X X X X X X X12B6H3 X X X X X X X X 12B6H4 X X X X X X X X

Expressions were performed as described in Example 2. Expression yieldsare summarized in Table 25.

The stability of the humanised nanobodies was measured as described inExample 7. Table 26 summarizes the OD280 nm concentrations (μg/ml) ofthe supernatants expressed as a percentage of the concentration at roomtemperature.

FIG. 16 shows the binding of humanised 12B6 nanobodies to vWF in ELISAperformed as described in Example 3.

The affinity of humanised 12B6 nanobodies for vWF was determined inBIACORE. KD values are summarized in Table 27.

Example 15 Humanisation of 12A2 Nanobody

Table 28 SEQ ID Nos: 90 to 94 represents five humanised 12A2 nanobodies.Tables III and IV list the following amino acid changes that wereperformed to achieve these sequences. FIG. 17 represents the alignmentof the humanised sequences for 12A2.

TABLE III non-limiting humanizing substitutions: Q1E K3Q E5V A14P A17SR27F E44G D46E 12A2H1 X X X X X X X 12A2H3 X X X X X X X 12A2H4 X X X XX X X 12A2H11 X X X X X X X X 12A2H13 X X X X X X X X

TABLE IV R76N M77S N82bS K83R P84A Q108L 12A2H1 X X X 12A2H3 X X X X12A2H4 X X X X 12A2H11 X X X 12A2H13 X X X X X X

Expressions were performed as described in Example 2. Expression yieldsare summarized in Table 29.

Stability of the humanised nanobodies was measured as described inExample 7. Table 30 summarizes the OD280 nm concentrations (μg/ml) ofthe supernatants expressed as a percentage of the concentration at roomtemperature. All humanised 12A2 nanobodies are very stable upon heatingat increasing temperatures.

The ELISA of FIGS. 18 and 19 was performed as described in Example 3.12A2H1 and 12A2H4 bind very well to vWF in ELISA.

The nanobodies were tested in the flow chamber at a concentration of 0.7μg/ml and 1.5 μg/ml. The same donor was used for all the experiments.The experiment was performed as described in Example 4. Results aresummarized in Table 31 and 32.

The affinity of humanised 12A2 nanobodies for vWF was determined inBIACORE. KD values are summarized in Table 33.

Example 16 Humanisation of 12A5 Nanobody

Table 34 SEQ ID Nos: 95 to 97 represents three humanised 12A5nanobodies. Table V lists the amino acid changes that were performed toachieve these sequences. FIG. 20 represents the alignment of thehumanised sequences for 12A5.

TABLE V non-limiting humanizing substitutions: A1E L23A Q44G G73N P74AK83R P84A 12A5H1 X X X X X 12A5H2 X X X X X X 12A5H3 X X X X X X X

Expressions were performed as described above in Example 2. Expressionyields after TALON purification are summarized in Table 35 for eachhumanised 12A5 nanobody.

Stability of the humanised 12A5 nanobodies was measured as describedabove in Example 7. The OD280 of the supernatants was measured andexpressed as percentage of the OD280 at room temperature. The resultsare summarized in Table 36. All humanised 12A5 nanobodies are comparablystable upon heating at increasing temperatures to the wild type.

ELISA was performed as described in Example 3. FIG. 21 illustrates thebinding activity for vWF in ELISA.

The affinity of humanised 12A5 nanobodies for vWF was determined inBIACORE. KD values are summarized in Table 37.

Example 17 Bivalent Humanised Nanobodies

Three humanised nanobodies 12A2H1, 12A2H4 and 12B6H2 were selected forbivalent form with the 3a linker. The sequences of these 3 nanobodiesdiffer only by a few amino acids as shown in FIG. 22. Table 38 SEQ IDNos 98 to 100 lists the sequences of the bivalent nanobodies. Table 38SEQ ID Nos 101- to 106 lists the sequences of humanised bivalentnanobodies linked with the GS9 and GS30 linker, respectively.

Expressions were performed as described in Example 2. The nanobodiescontaining a (His)₆-tag were purified on a Nickel column (TALON,Clonetech) as described by the manufacturer. The tag-sequence isEQKLISEEDLNGAA_(HHHHHH). Nanobodies without tags were purified onprotein A. Expression yields were calculated and are summarized in Table39.

Stability of the humanised bivalent nanobodies was measured as describedin Example 7. OD280 of the supernatants was measured and expressed aspercentage of the OD280 at room temperature. The results are summarizedin Table 40.

The nanobodies (humanised but also wild type) were tested in the flowchamber at a concentration of 0.15 μg/ml, 0.3 μg/ml and 0.6 μg/ml. Thesame donor was used for all the experiments. The experiment wasperformed as described in Example 4. FIG. 23 shows the platelet adhesionat different concentrations of bivalent nanobodies. Table 41 listsplatelet adhesion of wild type and humanised bivalent nanobodies.

D. Effect of (Bivalent) Nanobody on Arterial Thrombosis in a BaboonFolts Model Example 18 Baboon FOLTS Model with ALX-0081

In this study the efficacy and safety of ALX-0081 was evaluated in aFolts thrombosis model in baboons.

Also, the efficacy and safety of ALX-0081 in a Folts thrombosis model inbaboons was compared to other drugs currently used in the clinic, suchas Reopro, Plavix, Aspegic, Heparin and Epinephrin. All these werediluted in 0.9% sodium chloride and administered as intravenous bolusinjections. This study was as well designed to determine the effectivedose for each of these compounds.

Finally, the efficacy in a Folts thrombosis model in baboons of acombination of drugs that is currently used in the clinic in apercutaneous coronary intervention (PCI) setting was tested: Aspegic,Heparin, and Plavix. We furthermore evaluated if ALX-0081 can improvethe efficacy of this combination when added on top.

We looked at safety parameters such as induction of bleeding, vWF andfactor VIII levels, and platelet count, PT, and aPTT.

Study Protocol

The study protocol that was applied is the original Folts model and somemodifications described below (Folts J D, et al, Circulation. 1976;54:365-370).

Healthy male or female baboons (Papio ursinus) were used. The animalswere 8-17 kg of weight and were disease-free for at least 2 weeks priorto use. The baboons were fed with dry standard food only. The baboonswere used at different time points. The weight of the baboons aresummarized in table 42 (efficacy study ALX-0081 and comparison withindividual drugs) and table 50 (efficacy of a combination of drugs andALX-0081 on top of this combination).

Animals were anaesthetized and body temperature is maintained at 37° C.with a heating table. A segment of a femoral artery was dissected freefrom surrounding tissue. A shunt was placed between the femoral vein andfemoral artery to obtain high shear rates. The mean and phasic bloodflow was recorded continuously throughout the experiment. Baseline flowwas recorded for 20 minutes. The proximal dissection site of the femoralartery was then injured by applying two overlapping occlusions of theartery for 1 second using a forceps. A clamp was placed over the injuredsite to create an external stenosis.

A gradual decline in blood flow due to platelet adhesion and aggregationwas observed. When flow was reduced to zero, blood flow was restored byopening the clamp to dislodge the platelet-rich thrombus. Thisrepetitive pattern of decreasing blood flow following mechanicalrestoration is referred to as cyclic flow reductions (CFRs). Additionalendothelial injury was repeated if needed to finally obtain stable CFRsin these baboons. The number of times the thrombus needed to bedislodged determines the number of CFRs. FIG. 24 illustrates blood flowpattern during the Folts model in baboons.

After a 30-minute control period of reproducible CFRs, the vehicle wasadministered as an internal control and CFRs were followed up for 30more minutes. After this period, test agents (saline (n=2), Reopro(n=3), Aspegic (n=3), Plavix (n=4), Heparin (n=3) or ALX-0081 Nanobody™(n=9)) were provided via an intravenous bolus injection (followed by acontinuous infusion for ALX-0081) and monitoring was continued up to 30minutes after drug administration. This procedure was repeated forseveral times with escalating doses of the test substance. Theanti-thrombotic effect was quantified by comparing the length of CFRsbefore and after drug administration. When full inhibition of CFRs wasobserved, a new injury was applied in order to confirm that theinhibition was an effect of the treatment but not of a natural healingphenomenon. At the end of the experiments, Epinephrin (2.2 μg/kg/min)was injected in order to distinguish between a weak and a stronginhibition of the CFRs. Indeed, it has been demonstrated before thatCFRs reappear in the presence of Epinephrin when aspirin (a weakanti-platelet drug) is used in the same model.

The setup of the experiment is illustrated FIG. 25.

The length of the CFRs, after each dose of test compound are summarizedin tables 43-48. Doses at which full inhibition of CFRs is obtained areshaded.

A representative read out of the blood flow during the Folts modelexperiments is shown in FIGS. 26-31.

The results demonstrate that CFRs can be obtained in the control animalsfor at least 3 hours, without the need for a new injury in between. Themean length of the CFRs is 2-5 minutes and there is no effect on thelength of the CFRs by injection of saline (FIG. 26, table 43).

Aspegic

Three animals were injected with Aspegic (injectable Aspirin) and lookedfor inhibition of CFRs. In the clinic, a bolus injection of 250 mg (±3-5mg/kg) is administered to the patient, just before the start of apercutaneous coronary intervention (PCI) procedure. In two animals(baboon 3 and 5) no inhibition of CFRs could be obtained at doses ashigh as 80 and 40 mg/kg Aspegic respectively (FIG. 27, table 44). Inbaboon 4, it was very difficult to establish a stable repetitive patternof CFRs in the control phase. After several new injuries were made (thisis at the time we injected saline), stable CFRs were obtained. Fullinhibition of CFRs was obtained at the dose of 5 mg/kg Aspegic, but athigher doses and upon new injury, the CFRs returned, although the meanlength of the CFRs was 3-4 times longer than before administration ofAspegic. After infusion of Epinephrin, the CFRs returned immediately andcompletely (table 44).

Heparin

Three animals were injected with unfractionated Heparin and looked forinhibition of CFRs. In the clinic, a bolus injection of 60-70 IU/kg isadministered to the patient, and the aPTT (activated partialthromboplastin time) is monitored every 30 minutes. Extra Heparin isadministered if the aPTT is <250 seconds. In baboons 7 and 8, noinhibition of the CFRs could be obtained even not at doses as high as240 IU/kg (FIG. 28, table 45). In baboon 6, full inhibition of the CFRswas obtained at the first dose of 15 IU/kg and at higher doses, but whenwe made a new injury the CFRs returned each time. At the highest dose of240 IU/kg, CFRs were inhibited even after a new injury, but the flow wasdecreasing and upon infusion of Epinephrine, the CFRs returnedimmediately.

Plavix

Four baboons were treated with Plavix and used for the Folts study. Weused Plavix as injectable drug by re-suspending tablets in methanol.Therefore, we were able to perform a dose escalation experiment as forthe other drugs. In patients, 300-600 mg Plavix is administered orally,inhibition of platelet aggregation can be seen 2 hours after single oraldoses of Plavix. Already at the 2.5 mg/kg final dose in the baboons, aneffect on the length of the CFRs could be demonstrated, but thisinhibitory effect started only 10 minutes after injection (FIG. 29,table 46). In baboon 12, full inhibition of the CFRs was obtained atthis dose of 2.5 mg/kg. In the other three baboons full inhibition ofCFRs was obtained at the 5 mg/kg final dose. CFRs remained inhibitedwhen a new injury was made, but returned after infusion of Epinephrin.When Epinephrin infusion was stopped, the CFRs remained for another 5minutes, but then again full inhibition was obtained (FIG. 29).

Reopro

Reopro was tested for efficacy in the Folts model in three baboons. Inthe clinic, patients receive a dose of 250 μg/kg followed by acontinuous infusion of 7.5 μg/kg/hour. This is also the dose which weneeded in baboons 13, 14 and 15 to obtain full inhibition of the CFRs(final dose of 170-420 μg/kg (FIG. 30, table 47). We administered to thebaboons a bolus injection only. When a new injury was applied, completeinhibition of the CFRs was retained and infusion of Epinephrin could notreverse this inhibition (FIG. 30).

ALX-0081

Nine baboons received ALX-0081 and were used in the Folts model. In allbaboons full inhibition of CFRs was obtained at the dose of 30 μg/kg+45μg/kg/hour (final dose of 43 μg/kg). In 2 baboons, full inhibition wasalready obtained at the 10 μg/kg+15 μg/kg/hour (baboons 17 and 22).Inhibition was retained upon a new injury and after infusion ofEpinephrin in all nine baboons (FIG. 31, table 48).

Aspegic-Heparin-Plavix-ALX-0081 (Asp/Hep/Plav/ALX) Combinations

Seven baboons received a bolus injection of 5 mg/kg Aspirin, 60 IU/kgHeparin and increasing doses of Plavix. Extra Heparin was administeredat different time points to sustain a certain level (aPTT should be atleast doubled versus control). CFRs were monitored for 30 minutes aftereach dose of test compounds. We started at a dose of 1 mg/kg Plavix andadded 1 mg/kg after 30 minutes. The anti-thrombotic effect wasquantified by comparing the length of CFRs before and after drugadministration. When full inhibition of CFRs was observed, a new injurywas applied. Epinephrin was injected and continued till the end of theexperiment. If CFRs did not return, a new injury was applied. After 2-3CFRs increasing doses of ALX-0081 were added: (1), 3, 10 or 30 μg/kg. Wewaited after each dose of ALX-0081 for 2 CFRs and increased the doseuntil full inhibition of CFRs was obtained. At full inhibition of theCFRs, continuous infusion was started of 1.5 times dose ALX-0081/kg/hourfor 30 minutes and epinephrine infusion was continued. A new injury wasapplied after 10-15 minutes. The specifications of the baboons that wereused in this study are represented in table 49.

The results from these studies for each test compound individually aresummarized in table 50. A clear superior anti-thrombotic effect in theFolts thrombosis baboon model is observed for ALX-0081 and Reopro whencompared to Aspirin, Heparin or Plavix: upon new injury and afterinfusion of Epinephrin, the CFRs do not return in the Folts model in theALX-0081 and Reopro treated baboons in contrast to the model in Aspegic,Heparin or Plavix treated animals. The dose of ALX-0081 required forfull inhibition of CFRs is approximately 10-fold lower than the doseneeded for Reopro. Therefore, it is concluded that ALX-0081 is morepotent than Reopro.

After administration of a combination of 5 mg/kg Aspirin, 60 IU/kgHeparin and increasing doses of Plavix, in all seven baboon fullinhibition of CFRs was obtained at this final dose. The dose of Plavixrequired for full inhibition is 2.5-fold lower than the dose needed whenPlavix alone is administered. For all baboons tested, CFRs did notreturn when a new injury was made (FIG. 32). However, upon injection ofEpinephrin, CFRs returned spontaneously in baboons 5, 8, 9 and 10 andupon a new injury in baboons 4, 6 and 7. Extra Heparin was injected atthe same time as epinephrin. After 2 CFRs, increasing doses of ALX-0081were administered while continuing the infusion of Epinephrin. The doseof ALX-0081 was increased from 1 over 3-10 to 30 μg/kg. When fullinhibition of CFRs was obtained, a continuous infusion of ALX-0081 wasstarted at 1.5 times the effective dose/kg/hour. In all seven baboons,full inhibition of the CFRs was obtained at the 30 μg/kg dose. Thiseffective dose of ALX-0081 is the same as required for completeinhibition of CFRs in the Folts model when ALX-0081 is administeredalone.

Therefore, we can conclude that the efficacy of ALX-0081 is notincreased by simultaneous infusion of Plavix, Heparin and Aspegic. Thisobservation is completely in line with our hypothesis: ALX-0081 inhibitsthe very first interaction between platelets and the exposed collagen inthe damaged arterial wall. Plavix and Aspegic on the other hand inhibitfurther downstream in the cascade leading to the development of athrombus. Therefore, Plavix and Aspirin do not contribute to betterefficacy as ALX-0081 interferes already with the first step in thrombusformation. Moreover, when a new injury was applied at the effective doseof ALX-0081, the CFRs did not return, demonstrating a potentantithrombotic effect of this Nanobody™. The results are summarized intable 51.

Measurements

The following parameters were measured: a) bleeding analysis, b) vWFconcentration, Factor VIII levels, and platelet count, PT and aPTT c)ristocetin-induced platelet aggregation d) ALX-0081 concentration, ande) analysis of arterial sections for restenosis, f) immunogenicity ofALX-0081

a) Bleeding Analysis

To analyze bleeding, an incision was made with a scalpel in the groin.This was done at 15 minutes after recording baseline flow, when theinjury was made in the artery. Gauzes were inserted in the wound andreplaced every 30 minutes just before each new dose of test compound.The amount of blood loss after each dose of test compound was determinedby weighing the gauzes. Blood loss is expressed relative to the amountof blood loss in the second control gauze (during the saline injection)(tables 52-54).

For all baboons treated with Plavix and Reopro, blood loss is high (upto 9-40 fold respectively at the highest dose), starting from theeffective dose on. For animals treated with ALX-0081 bleeding is lowerthan in the Plavix treated animals, and much lower when compared toReopro treated animals.

In order to determine the safety versus efficacy level of Plavix, Reoproand ALX-0081 as antithrombotic drugs, the averages of blood lossrelative to the second control gauze are shown for these drugs infunction of the drug dose as multiple of the effective dose (FIG. 33).The effective dose for Plavix is 5 mg/kg, for Reopro 250 μg/kg and forALX-0081 30 μg/kg (table 46-48). These results nicely demonstrate thesuperior safety of ALX-0081 when compared to Reopro and Plavix: thewindow in which ALX-0081 could be administered without a major increasein bleeding is much wider compared to Plavix and Reopro.

The results from the averages of blood loss in the gauzes (if available)were combined with the averages of the lengths of the CFRs in the FIGS.34-36.

A broad therapeutic window was observed for ALX-0081 in the Folts model:a strong antithrombotic effect could be demonstrated without any majorbleedings for cumulative doses ranging from 43 μg/kg up to 403 μg/kg(FIG. 36). In contrast however, the therapeutic window for Reopro andPlavix in the same model was much more narrow compared to ALX-0081,combining an effective antithrombotic effect with a high blood loss(FIG. 34-35).

The average of the total amount of blood loss (=sum of blood loss fromthe gauzes of the first five doses of test compound) as relative to thesecond control gauze are summarized in table 55. In this table we alsoindicate the final dose as multiple of the effective dose (=sum of thefive doses divided by the effective dose). As mentioned before, theeffective dose for Plavix is 5 mg/kg, for Reopro 250 μg/kg and forALX-0081 30 μg/kg.

The results shown in table 55 clearly show that total blood loss issignificantly increased in the animals treated with Plavix and to aneven higher extent in the animals treated with Reopro. Blood loss inanimals receiving ALX-0081 is 2-fold and 4 fold less than in Plavix orReopro treated animals, respectively. This again clearly demonstratesthat ALX-0081 is safer than Plavix and Reopro in terms of bleeding risk,although doses of more than 10-fold the effective dose were used.

The effective combination of Aspegic, Heparin and Plavix results in anincrease in blood loss of up to 14 fold when compared to the controlgauze (table 56). Addition of ALX-0081 on top of the combination ofAspegic, Heparin and Plavix does not result in increased bleeding exceptfor baboons 4 and 7. In baboon 7, bleeding was much increased afteradministration of epinephrine, extra heparin and non effective doses ofALX-0081, but was lower again after administration of the effective doseof ALX-0081. These results demonstrated that ALX-0081 is safe when addedon top of the combination of drugs that is currently used in a clinicalsetting.

b) vWF Concentration, Factor VIII Level, Platelet Count, PT and aPTT

vWF

The vWF levels in the platelet rich plasma (PRP) of blood samples takenafter administration of the different doses of the drugs in the Foltsmodel were determined using an immunosorbent assay and expressed as apercentage of the human standard (WHO 5^(th) International Standard forfactor VIII and VWF)

The results clearly demonstrate that the different drugs used in themodel have no major effect on the vWF level.

Factor VIII

The factor VIII levels in the PRP of blood samples taken afteradministration of the different doses of the drugs in the Folts modelwere determined using the aPTT test. We did not test the plasma samplesof the baboons treated with Heparin, as we demonstrated that Heparinprolongs the aPTT time. The FVIII levels were expressed as percentage ofthe first control sample, taken 10 minutes after injury of the femoralartery. We do not see any effect of the treatments on the aPTT test.

Platelet Count, PT and aPTT

The platelet count measurements during the Folts model experiments wereperformed. The data showed that baboon 15 has a very low platelet countwhen compared to the other animals. The platelet counts for all kind oftreatments, except for the Plavix treatment, are very comparable to whatwe see in the control animals and are fairly constant over time.

The PT values demonstrate no effect of the test compounds on the PTtime, except for baboons treated with the 240 IU/kg dose of Heparinwhere a minor increase in PT was observed.

The aPTT values observed during the Folts model studies are summarized.These results indicate that the test compounds have no effect on theaPTT values, except for the baboons treated with Heparin. In theseanimals, aPTT values are prolonged from the 30-60 IU/kg dose on, as isalso observed in patients. Heparin acts as an anticoagulant by forming acomplex with antithrombin and catalyzing the inhibition of activatedblood coagulation factors such as XIa, IXa, Xa and thrombin (factorIIa). These factors are all involved in the intrinsic coagulationcascade of which its functionality is measured in the aPTT test.

c) Measurement of Ristocetin-Induced Platelet Aggregation in BloodObtained from Baboons Treated with ALX-0081

Blood obtained from baboons treated with ALX-0081 was analyzed forinhibition of platelet aggregation. Platelet aggregations were performedon a Chronolog whole blood and optical Aggregometer (Model 560CA,Chronolog, USA). PRP was prepared (collected on 0.38 mol/L citrate), bycentrifuging the whole blood at 1200 rpm for 5 minutes. The upperfraction containing the PRP was carefully removed. The lower fractionwas further centrifuged at 3000 rpm for 10 minutes to prepare plateletpoor plasma (PPP). Platelets were counted in PRP and diluted in PPP to afinal concentration of 200.000 platelets per microliter. 3 mg/mlristocetin (DAKO) was added and aggregation was measured.

The ex vivo platelet aggregation is measured in the blood samples takenduring the Folts experiment in the baboons treated with ALX-0081. TheGPIb-IX-V dependent platelet aggregation through vWF is measured usingristocetin as a modulator. The % aggregation is measured at each timepoint and at each dose. The control sample is taken at 10 minutes afterarterial injury.

Results from the RIPA test are compared to the inhibition of the CFRsfor each baboon treated with ALX-0081 (FIG. 37). As shown in FIG. 37, aninverse relation between the RIPA and the length of the CFRs isobserved. Moreover, these results demonstrate that full inhibition inthe RIPA test is obtained at lower doses than full inhibition of CFRs inbaboons 16, 18, 19 and 23. For baboons 20, 21, 22 and 24 the resultswith RIPA compare very well with the results for efficacy in the Foltsmodel.

d) Concentration of ALX-0081

Microtiterplates are coated with mouse polyclonal anti-myc overnight at4° C. at a 1000-fold dilution. The plates are washed with PBS-Tween andblocked for 2 hours at RT with PBS-1% casein. Samples are diluted in anon-coated microtiterplate in 25% reference baboon plasma. The standardcurve is prepared by diluting the nanobody in the same reference baboonplasma sample. Samples are applied on the anti-myc coated plates andallowed to bind for 2 hours at RT. The plates are washed 5 times withPBS-Tween. Rabbit anti-vWF-HRP (DAKO) is applied at a 3000-fold dilutionfor one hour at RT. For measuring OD405 nm, samples are washed 5 timeswith PBS-Tween and ABTS/H₂O₂ substrate is added.

We determined the concentration of ALX-0081 in plasma samples taken at10 minutes after each bolus injection. The bolus injection wasimmediately followed by a continuous infusion. The concentrations(μg/ml) are summarized in table 58.

For all baboons an increasing level of ALX-0081 in the plasma sampleswas measured by ELISA after dose-escalation of ALX-0081. For baboon 16,consistently higher amounts of ALX-0081 were determined in the plasmasample taken after the 10 μg/kg dose compared to the sample taken after30 μg/kg. The ALX-0081 level in that sample is also substantially higherthan what is noted for all other baboons given the same dosing scheduleof ALX-0081. As the levels of ALX-0081 for baboon 16 after the higherdoses are in line of the expectations, we assume that an unknownabnormality during blood sampling accounts for this outlier.

The concentration of ALX-0081 for each dose in the different baboons isvariable. For the 3 μg/kg dose, the concentration ranges between 0.03and 0.14 μg/ml for 10 μg/kg between 0.18 and 1.23 μg/ml, for 30 μg/kgbetween 0.51 and 1.14 μg/ml, for 90 μg/kg between 1.38 and 6.77 μg/mland for 270 μg/kg between 4.03 and 35.14 μg/ml.

In FIG. 38, we plot the concentration of ALX-0081 in plasma versus thelength of the CFRs.

The concentration of ALX-0081 required for full inhibition of CFRs isbetween 0.3 and 0.5 μg/ml, which is in full agreement with theconcentration required to inhibit platelet adhesion to collagen in theflow chamber at high shear rate, when ALX-0081 is spiked in human blood.In blue (FIG. 38, panel B) we indicated the concentration range ofALX-0081 where inhibition starts (leaving out the 10 μg/kg dose inbaboon 16).

When we plot the concentration of ALX-0081 versus the relative amount ofblood loss from the gauzes, we observe a more than 2-fold increase inbleeding at doses above 1 μg/ml (FIG. 39). A 10-fold increase in bloodloss was observed when the ALX-0081 concentration is 19 μg/ml, which is40-60-fold the effective concentration.

e) Analysis of Arterial Sections for Restenosis

Four weeks after treatment of the second artery, the arteries aredissected free from surrounding tissue. The artery is tied at the upperand low site of the endothelial injury including the site where theshunt was placed. A section is removed of 2 centimeter, cut in a lower(shunt site) and upper part (stenosed and injured site) and stored in10% formaldehyde. The baboons are then sacrificed by euthanasiainjection. The arteries are marked according to origin and cut intorings of 2 mm each. The rings are placed into marked cassettes suitablefor histology processing. The cassettes are then placed overnight in anautomated VIP Tissue Tek processor following the overnight processingschedule as described in Bancroft (Bancroft, John D., Stevens Alan(1990). Theory and Practice of Histological Techniques. Third Edition).

After processing, the arteries are embedded in marked paraffin waxblocks and cooled on a freeze plate. The wax blocks are cut in seriessections of 4 micron each on a rotary microtome. Sections are picked upon glass slides and stained for histological evaluation. Haematoxylinand Eosin as well as Verhoeff's method for elastic fibers stains areperformed on each of the arteries (Bancroft, John D., Stevens Alan(1990). Theory and Practice of Histological Techniques. Third Edition).After staining, the slides are dehydrated, cleared, mounted andlabelled. Blind analysis of the sections is performed.

f) Immunogenicity Analysis

The presence of ALX-0081 immunoglobulins in plasma of three baboons wasevaluated by two methods, respectively an ELISA method and a SPR-basedmethod on Biacore. The baboons were treated for 8 weeks with increasingdoses of ALX-0081 (starting from 10 μg/kg). During said 8 weeks, noimmunogenic response could be observed upon injection of ALX-0081. Thehalf-life of ALX-0081 ranged between 7 and 9 hours.

Example 20 Use of vWF as an Antidote for ALX-0081

Despite the proven safety of ALX-0081 in baboons, and the rapidclearance of the Nanobody™, we decided to evaluate the use of vWF as anantidote for ALX-0081. This was tested in a Folts model in baboons wherewe evaluated if the inhibitory effect of ALX-0081 on arterial thrombusformation can be reversed by injection of vWF.

The experimental procedure followed the original Folts model with themodifications as described in the previous example 18.

Healthy male baboons (Papio ursinus) were used in this study. Theanimals were 9-12 kg of weight and were disease-free for at least 2weeks prior to use. The baboons were fed with dry standard food only.

Three baboons were used in this study, the length of the CFRs during thecontrol phase, after administration saline, ALX-0081 and vWF issummarized in table 59.

The blood flow as a function of time for each baboon and experiment isshown in FIG. 40.

In all three baboons, full inhibition of CFRs was obtained at the 30μg/kg+45 μg/kg/hour dose of ALX-0081, even when a new injury wasapplied, the CFRs did not return. Upon injection of the first dose ofvWF (250 IU), the flow gradually decreased, but CFRs did not returnuntil an extra dose of 250 IU of vWF was administered. This resultdemonstrated nicely that the activity of ALX-0081 can be reversed byadministration of vWF, and that therefore, vWF would be a good antidotefor this Nanobody™.

Thus, another aspect of the invention relates to the use of vWF, of asuitable fragment thereof, of DDAVP (desmopressinor) or a suitablefragment thereof, or of a pharmaceutical composition comprising any ofthe foregoing, as an antidote for complications or undesired sideeffects associated with the use of a Nanobody, protein or polypeptideagainst vWF, in particular a Nanobody, protein or polypeptide asdescribed herein.

Example 21 Effects of ALX-0081 on Platelet Adhesion to EndothelialCell-Derived UlvWF and on the Activity of ADAMTS-13

This study serves as a proof of concept for the use of ALX-0081 as adrug in TTP patients. Perfusions of platelets reconstituted in TTPplasma (no ADAMTS13) are performed on endothelial cells secreting ULvWF,in the absence and presence of ALX-0081. In a separate experiment wetest if ALX-0081, which binds to the A1 domain of vWF, interferes withthe ADAMTS-13 activity. ADAMTS-13 binds to and cleaves the A2 domain ofvWF.

Endothelial cells were obtained from human umbilical cord veins by themethod of Maruyama (Z. Zellforsch. Mikrosk. A4nat. 60:69; 1963).Endothelial cells were activated with 100 μM histamine (Sigma-Aldrich,St Louis, Mo.) for 15 minutes at room temperature before the perfusionexperiments.

Blood was drawn from healthy volunteers who denied ingestion of aspirinor other nonsteroidal anti-inflammatory drugs (NSAIDs) for the preceding10 days into one-tenth volume 3.4% sodium citrate. Platelet-rich plasma(PRP) was prepared from whole blood by centrifugation (10 minutes at 200g at room temperature). The PRP was acidified by addition of one-tenthvolume of ACD (2.5% trisodium citrate, 1.5% citric acid, and 2%D-glucose), and the platelets were spun down (500 g, 15 minutes). Theplatelet pellet was resuspended in HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)-Tyrode buffer (10mM HEPES, 137 mM NaCl, 2.68 mM KCl, 0.42 mM NaH₂PO₄, 1.7 mM MgCl₂, 5 mMD-glucose, pH 6.5). Prostacyclin (PGI₂, 10 ng/mL) was added to preventplatelet activation during the subsequent washing step. Platelets werespun down and resuspended in a small volume of HEPES-Tyrode buffer. Thisplatelet suspension was diluted in HEPES buffer at pH 7.4, or in TTPplasma.

Perfusions were performed in a single-pass perfusion chamber asdescribed previously. The experiment was followed by real-timevideomicroscopy.

In a second type of experiment, different reaction mixtures wereprepared as summarized in table 60, however, without the addition of theA1A2A3 construct. The A1A2A3 construct is a recombinant fragmentconsisting out of the A1, A2 and A3 domain of vWF. Mixtures werepre-incubated for 5 minutes at 37° C. after which the A1A2A3 fragment isadded and the mixture is incubated in a waterbath overnight at 37° C.The next day a reducing SDS-PAGE is run on the samples (12%, and usingas marker the Precision Plus Protein Standards from BioRad) and blottedon Immobilon-FL (Millipore). The blot was blocked for 2 hours at roomtemperature with blockbuffer (1:1 Odyssey blockbuffer in 1×TBS pH=7.4)and incubated with Rabbit polyclonal anti-vWF (DAKO). Alexa Fluor 680goat anti-rabbit was used for detection. Scanning was done on theODYSSEY to detect the degradation products.

Control Experiment: Binding of Platelets to ULvWF

Endothelial cells were isolated from freshly obtained human umbilicalcords by collagenase digestion of the interior of the umbilical vein.The cells were grown in tissue culture as a homogeneous population.Cultured human endothelial cells grow as monolayers of closely opposed,polygonal large cells and they contain cytoplasmic inclusions(Weibel-Palade bodies). Histamine-stimulated endothelial cells isolatedfrom human umbilical cords express ultra-large von Willebrand factor(ULvWF) on their surface. Perfusion of these stimulated cells withisolated blood platelets, which are suspended into either buffer orplasma from a patient with acquired TTP, results in deposition ofplatelets onto the ULvWF (8). These ULvWF-adhered platelets appear asso-called ‘strings’, which are visible when the perfusion experiment ismonitored by real-time video microscopy (FIG. 41).

Inhibition by ALX-0081 for the Generation of Platelet Strings

Platelets were resuspended in buffer or in TTP plasma and theconcentrations of ALX-0081 used in this experiment were 0.2, 2 and 10μg/ml. Histamine-stimulated endothelial cells isolated from humanumbilical cords are perfused with these platelet suspensions asdescribed above.

Addition of ALX-0081 to platelets resuspended in buffer or in plasmafrom a TTP patient results in a complete inhibition of string formationunder all conditions tested (FIG. 42). Perfusion experiments wereperformed at a shear stress of 2.5 dyn/cm2 for 4 minutes. During this 4minute perfusion at least 20 microscopic fields were examined, and inthe presence of the Nanobody™, no strings could be demonstrated at allconditions tested (FIG. 42).

Cleavage of ULvWF by ADAMTS-13

ADAMTS-13 reduces the size of large and ultralarge VWF multimers tosmaller forms by specifically cleaving the Y842/M843 peptide bond in theVWF A2 domain. Two types of assays were used to evaluate the effect ofALX-0081 on the cleavage of ULvWF by ADAMTS-13: i.e. a perfusion assayand an assay observing the cleavage of a recombinant vWF fragment.

In a first experiment, strings were generated by a 4 minute perfusion ofwashed platelets resuspended in buffer over histamine-stimulatedendothelial cells. Subsequently, the non-adhered platelets were washedaway by a 4 minute perfusion of buffer. After that, buffer was perfusedfor an other 4 minutes, followed by a 4 minute perfusion of poolednormal plasma containing ADAMTS-13. Detachment of the platelet stringswas observed after perfusion of pooled normal plasma (FIG. 43). Morethan 95% of the strings were cleaved after the 4 minute perfusion.

In a next experiment, strings were generated by a 4 minute perfusion ofwashed platelets resuspended in buffer over histamine-stimulatedendothelial cells and the non-adhered platelets were washed away by a 4minute perfusion of buffer as above. After that, ALX-0081 (10 μg/ml inbuffer) was perfused for 4 minutes, followed by a 4 minute perfusion ofpooled normal plasma containing ALX-0081 (10 μg/ml=10-fold molar excessover vWF) and ADAMTS-13. We could clearly demonstrate detachment of theplatelet strings and 95% of the strings were cleaved after the 4 minuteperfusion (FIG. 44)

These results clearly demonstrate that ALX-0081 does not have an effecton the cleavage of ULvWF strings by ADAMTS-13.

In a second assay, a recombinant fragment containing the A1-A2-A3 domainof vWF was mixed with normal pool plasma (NPP) containing ADAMTS-13,resulting in proteolytic cleavage of the fragment which was observed bya Western Blot analysis. ADAMTS-13 activity was tested in the absenceand presence of 10 μg/ml ALX-0081. As indicated in FIG. 45 ALX-0081 hasno effect on the cleavage of the vWF fragment (lanes 6-7-8).

In order to demonstrate that the observed cleavage is specific forADAMTS-13, a control experiment in the presence of EDTA was performed asEDTA inhibits the activity of ADAMTS-13. As expected, the presence ofEDTA in NPP resulted in inhibition of cleavage of the fragment (FIG. 45lane 4).

This experiment again proves that ALX-0081 has no effect on theADAMTS-13 activity.

TABLE 8 Sequence listing of anti-vWF nanobodies SEQ Name ID NO Sequence12A5 60 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQ VTVSS 12B1 61QVQLVESGGGLVQAGGSLRLSCAASGRTFSNYGMGWFRQAPGKEREFVTSISWSGTYTAYSDNVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCAAQSRYRSNYYDHDDKYAYWG QGTQVTVSS 12B6 62QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNF WGQGTQVTVSS 12D11 63AVQLVDSGGGLVQAGGSLRLSCTASERTTFSSYTLGWFRQAPGKEREFVGGISWSGVSTDYAEFAKGRFTISRDHAANTVYLEMNSLKPEDTAVYYCAALGRYRSNWRNIGQYDYWG QGTQVTVSS 12E3 64EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYGMGWFRQAPGKEREFVTSISWSGSYTAYADNVKGRFTISRDNAKNTVYLQMDSLKPGDTAVYYCAAQSRYSSNYYDHDDKYAYWG QGTQVTVSS 12C9 65AVQLVESGGGLVQPGGSLKLSCATSGSIFSSSAMAWYRQASGKQRELVATITSGGRTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYDCNFVVDGKRAPWGQGTQVTVSS 14F8 66AVQLVESGGGLVQAGESLRLSCTSSGRAFSYYNTGWFRQAPGKEREFVAAISWSGGLTYYADSVKGRFTISRDNAKDMVYLQMASLKPEDTAVYYCAANRRQKTVQMGERAYDYWGQ GTQVTVSS

TABLE 9 Expression yields of anti-vWF nanobodies Nanobody Yield (mg/l)after TALON 12A5 13 12B1 6 12B6 16 12D11 8 12E3 4 12C9 25 14F8 48

TABLE 10 Platelet adhesion in perfusion chamber of anti-vWF nanobodiesNanobody Control % Platelet adhesion at 2 μg/ml 12A5 60 ± 7 10 ± 3 12B160 ± 7 56 ± 3 12B6 60 ± 7 19 ± 5 12D11 60 ± 7 61 ± 7 12E3 60 ± 7 54 ± 112C9 71 ± 3 68 ± 8 14F8 71 ± 3  51 ± 10

TABLE 11 Sequence listing of 12B6 and 12A5 homologue nanobodies SEQ NameID NO Sequence 12A5 homologue sequences 12B4 67QVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGLYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQ VTVSS 12E8 68AVQLEESGGGLVQPGGSLRLSCLASGRIFSIGAMGLYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCYANLKQCDYGYRFNDYWGQGTQ VTVSS 12A6 69QVQLVESGGGLVQPGGSLRLSCLASGRIFSIGTMGLYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDGAKNTVYLQMNSLRPEDTAVYYCYANLKQGDYGYRFNDYWGQGTQ VTVSS 12D8 70AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGTMGLYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDGAKNTVYLQMNSLRPEDTAVYYCYANLKQGDYGYRFNDYWGQGTQ VTVSS 12B6 homologue sequences12A2 71 QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTQVTVSS 12F2 72QVKLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGRERDVVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRSLPSEYTF WGQGTQVTVSS 14H10 73QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLEMNNLKPDDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTQVTVSS

TABLE 12 Estimated K-on, K-off and KD values for 12A5 homologuenanobodies Nanobody Koff (×10⁻³/s) Kon (×10⁶ 1/Ms) KD (nM) 12A5 2.510.629 3.98 12B4 2.2 0.544 4.05 12E8 2.93 0.171 17.1 12A6 4.72 0.188 25.112D8 5.84 0.139 41.9

TABLE 13 Estimated K-on, K-off and KD values for 12B6 homologuenanobodies Nanobody Koff (×10⁻³/s) Kon (×10⁶ 1/Ms) KD (nM) 12B6 5.972.55 2.33 12A2 3.49 1.11 3.13 12F2 4.04 6.41 6.3 14H10 3.97 6.84 5.81

TABLE 14 Real KD value of 12B6, 12A2 and 12A5 nanobodies Nanobody Koff(×10⁻³/s) Kon (×10⁶ 1/Ms) KD (nM) 12B6 10.03 2.28 4.5 12A2 9.9 2.24 4.412A5 3.3 1.22 2.7

TABLE 15 Platelet adhesion in perfusion chamber of 12B6, 12A2 and 12A5nanobodies % Platelet adhesion Nanobody 0 μg/ml 0.2 μg/ml 0.4 μg/ml 0.6μg/ml Control 71 ± 3 — — — 12B6 — 59 ± 6 43 ± 7  27 ± 8 12A2 — 58 ± 8 40± 10 11 ± 8 12A5 — 50 ± 7 27 ± 10  2 ± 2

TABLE 16 Concentration of 12B6, 12A2 and 12A5 nanobodies after heatingat increasing temperatures Nano- body RT 37° C. 50° C. 60° C. 70° C. 80°C. 90° C. 12B6 100 97 100 104 94 91 68 12A2 100 106 104 100 93 87 9012A5 100 108 107 98 83 75 66

TABLE 17 Sequence listing of bivalent nanobodies SEQ ID Name NO Sequence12A2-3a-12A2 74 QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSV EGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS AAAEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYP DSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVT VSS 12A2-GS9-12A2 75QVKLEESGGGLVQAGGALRLSCAASGRTFSYN PMGWFRQAFGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYC AAAGVRAEDGRVRTLPSEYTFNGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGALRLSCA ASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLK PEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A2-G530-12A2 76 QVKLEESGGCLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSV EGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPM GWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEGTAVYYCAA AGVRAEDGRVRTLPSEYTFWGQGTQVTVSS12A5-3a-12A5 77 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVK GRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSAAAEVQ LVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRF TISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12A5-G59-12A5 78AVQLVESGGGLVQPGGSLRLSCLASGRIFSIG AMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCY ANLKQGSYGYRFNDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCLASGRIF SIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVY YCYANLKQGSYGYRFNDYWGQGTQVTVSS12A5-G530-12A5 79 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVK GRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQA PGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSY GYRFNDYWGQGTQVTVSS 12B6-3a-12B6 80QVQLVESGGGLVQAGGALRLSCAASGRTFSYN PMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYC AAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSAAAEVQLVESGGGLVQAGGALRLSCAASGRTF SYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAV YYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVT VSS12B6-G59-12B6 81 QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSV EGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTG GSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWG QGTQVTVSS 12B6-G530-12B6 82QVQLVESGGGLVQAGGALRLSCAASGRTFSYN PMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYC AAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV QLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEG RFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS

TABLE 18 Sequence listing of linker sequences SEQ Name ID NO Sequence 3a83 AAA GS9 84 GGGGSGGGS GS30 85 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

TABLE 19 Expression yields of bivalent 12B6, 12A2 and 12A5 nanobodiesYield (mg/l) after TALON or Nanobody other purification 12B6 1612B6-3a-12B6 9 12B6-GS9-12B6 16 12B6-GS30-12B6 17 12A2 18 12A2-3a-12A245 12A2-GS9-12A2 22 12A2-GS30-12A2 11 12A5 13 12A5-3a-12A5 1012A5-GS9-12A5 11 12A5-GS30-12A5 18

TABLE 20 Concentration of 12B6 bivalent nanobodies after heating atincreasing temperatures Nano- body RT 37° C. 50° C. 60° C. 70° C. 80° C.90° C. 12B6 100 97 100 104 94 91 68 12B6- 100 103 96 87 9 8 6 3a- 12B612B6- 100 103 94 88 19 8 7 GS9- 12B6 12B6- 100 100 100 98 46 14 11 GS30-12B6

TABLE 21 Concentration of 12A2 bivalent nanobodies after heating atincreasing temperatures Nano- body RT 37° C. 50° C. 60° C. 70° C. 80° C.90° C. 12A2 100 106 104 100 93 87 90 12A2- 100 87 88 91 55 50 43 3a-12A2 12A2- 100 102 113 138 91 13 15 GS9- 12A2 12A2- 100 115 93 116 81 4934 GS30- 12A2

TABLE 22 Concentration of 12A5 bivalent nanobodies after heating atincreasing temperatures 37° 50° 60° 70° Nanobody RT C. C. C. C. 80° C.90° C. 12A5 100 108 107 98 83 75 66 12A5-3a-12A5 100 101 114 29 6 4 612A5-GS9-12A5 100 104 115 32 13 14 10 12A5-GS30-12A5 100 104 87 7 6 3521

TABLE 23 Platelet adhesion in perfusion chamber of 12A2 bivalentnanobodies % Platelet adhesion Nanobody 0 μg/ml 0.1 μg/ml 0.2 μg/ml 0.4μg/ml Control 81 ± 5 — — — 12A2 — 78 ± 2 72 ± 6 61 ± 8 12A2-3a-12A2 — 74± 5 50 ± 3 33 ± 0 12A2-GS9-12A2 — 81 ± 1 73 ± 1 40 ± 2 12A2-GS30-12A2 —81 ± 3 73 ± 3 37 ± 1

TABLE 24 Sequence listing of humanised 12B6 nanobodies SEQ ID Name NOSequence 12B6H1 86 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQ VTVSS 12B6H2 87EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQ VTVSS 12B6H3 88EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRDVVAAISRTGGSTYYARSVEGRFTISRDNAKNMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQ VTVSS 12B6H4 89EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRDVVAATSRTGGSTYYARSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQ VTVSS

TABLE 25 Expression yields of wild type and humanised 12B6 nanobodiesYield (mg/l) after TALON or other Nanobody purification 12B6 16 12B6H1 312B6H2 9 12B6H3 8 12B6H4 3

TABLE 26 Concentration of wild type and humanised 12B6 nanobodies afterheating at increasing temperatures Nano- body RT 37° C. 50° C. 60° C.70° C. 80° C. 90° C. 12B6 100 97 100 104 94 91 68 12B6H1 100 101 100 9745 58 54 12B6H2 100 97 96 96 83 46 53 12B6H3 100 101 98 97 74 73 6512B6H4 100 101 100 93 41 66 54

TABLE 27 KD values for wild type and humanised 12B6 nanobodies NanobodyKD (nM) 12B6 4.4 12B6H1 4.4 12B6H2 3.5 12B6H3 9 12B6H4 7.3

TABLE 28 Sequence listing of humanised 12A2 nanobodies SEQ ID Name NOSequence 12A2H1 90 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTQVTVSS 12A2H3 91EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKNMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTQVTVSS 12A2H4 92EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTQVTVSS 12A2H11 93EVQLVESGGGLVQPGGSLRLSCAASGFTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRNVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTQVTVSS 12A2H13 94EVQLVESGGGLVQPGGSLRLSCAASGFTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTF WGQGTLVTVSS

TABLE 29 Expression yields of wild type and humanised 12A2 nanobodiesYield (mg/l) after TALON or other Nanobody purification 12A2 18 12A2H111 12A2H3 11 12A2H4 11 12A2H11 15 12A2H13 11

TABLE 30 Concentration of wild type and humanised 12A2 nanobodies afterheating at increasing temperatures Nano- 37° body RT C. 50° C. 60° C.70° C. 80° C. 90° C. 12A2 100 106 104 100 93 87 90 12A2H1 100 99 99 99100 89 80 12A2H3 100 102 101 102 102 90 89 12A2H4 100 100 101 100 99 9083 12A2H11 100 111 113 107 103 85 67 12A2H13 100 104 103 103 100 90 81

TABLE 31 Platelet adhesion of wild type and humanised 12A2 nanobodies inperfusion chamber at 0.7 and 1.5 ug/ml % Platelet adhesion Nanobody 0μg/ml 0.7 μg/ml 1.5 μg/ml Control 73 ± 4 — — 12A2 — 36 ± 3 34 ± 5 12A2H1— 49 ± 1 47 ± 3 12A2H3 — 62 ± 4 63 ± 1 12A2H4 — 55 ± 1 54 ± 1 12A2H11 —57 ± 1 52 ± 1 12A2H13 — 67 ± 4 67 ± 4

TABLE 32 Platelet adhesion of wild type and humanised 12A2 nanobodies inperfusion chamber at 0.5, 1 and 2 ug/ml % Platelet adhesion Nanobody 0μg/ml 0.5 μg/ml 1 μg/ml 2 μg/ml Control 72 ± 1 — — — 12A2 —  33 ± 10  35± 11  10 ± 10 12A2H1 — 40 ± 9 43 ± 3 38 ± 5 12A2H4 — 61 ± 1 57 ± 1 46 ±5

TABLE 33 KD values for wild type and humanised 12A2 nanobodies NanobodyKD (nM) 12A2 3.1 12A2 H3 14.6 12A2 H11 10.6 12A2 H13 38.8

TABLE 34 Sequence listing of humanised 12A5 nanobodies SEQ ID Name NOSequence 12A5H1 95 EVQLVESGGGLVQPGGSLRLSCAASGRIFSIGAMGMYRQAPGKGRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLRAEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12A5H2 96EVQLVESCGCLVQPGGSLRLSCAASCRIFSIGAMCMYRQAPGRGRELVATITSGGSTNYADPVKGRFTISRDGAKNTVYLQMNSLRAEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12A5H3 97EVQLVESGGGLVQPGGSLRLSCAASGRIFSIGAMGMYRQAPGKGRELVATITSGGSTNYADPVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS

TABLE 35 Expression yields of wild type and humanised 12A5 nanobodiesNanobody Yield (mg/l) after TALON 12A5 13 12A5H1 8 12A5H2 9 12A5H3 11

TABLE 36 Concentration of wild type and humanised 12A5 nanobodies afterheating at increasing temperatures Nanobody 37° C. 50° C. 60° C. 70° C.80° C. 90° C. 12A5 108 107 98 83 75 66 12A5H1 99 91 86 60 69 63 12A5H299 108 90 58 67 60 12A5H3 101 97 97 67 73 64

TABLE 37 KD values for wild type and humanised 12A5 nanobodies NanobodyKD (nM) 12A5 1.6 12A5H1 1.8 12A5H2 12.8  12A5H3 ND

TABLE 38 Sequence listing of humanised bivalent nanobodies SEQ ID NameNO Sequence 12A2H1-3a-12A2H1 98 EVQLVESGGGLVQPGGSLRLSCAASGRTESYNPMGWFRQAPGKGRELVAAISRTGGST YYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYT FWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGK GRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGV RAEDGRVRTLPSEYTFWGQGTQVTVSS12A2H4-3a-12A254 99 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGST YYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYT FWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGK GRELVAAISRTGGSTYYFDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGV RAEDGRVRTLPSEYTFWGQGTQVTVSS12B6H2-3a-12B6H2 100 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGST YYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYN FWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNFMGWFRQAPGK GREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGV RAEDGRVRTLPSEYNFWGQGTQVTVSS12A2H1-G59-12A2H1 101 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGST YYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYT FWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWF RQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYY CAAAGVRAEDGRVRTLPSEYTFWGQGTQV TVSS12A2H4-GS9-12A2H4 102 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGST YYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYT FWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWF RQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYY CAAAGVRAEDGRVRTLPSEYTFWGQGTQV TVSS1256H2-G59-12B6H2 103 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGST YYARSVEGRFTISRDNAKRMVYLQNNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSSYN FWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWF RQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYY CAAAGVRAEDGRVRTLPSEYNFWGQGTQV TVSS12A2H1-G530-12A2H1 104 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGST YYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYT FWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG SLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDN AKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A2H4-GS30-12A2H4 105EVQLVESGGGLVQPGGSLRLSCAASGRTF SYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLR AEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGR ELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRASDTAVYYCAAAGVRA EDGRVRTLPSEYTFWGQGTQVTVSS12E6H2-G530-12B6H2 106 EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGST YYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYN FWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG SLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDN AKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS

TABLE 39 Expression yields of humanised bivalent nanobodies NanobodyTags Yield (mg/l) 12A2H1-3a-12A2H1 Yes 5 12A2H1-3a-12A2H1 No 612A2H4-3a-12A2H4 Yes 10 12A2H4-3a-12A2H4 No 7 12B6H2-3a-12B6H2 Yes 1012B6H2-3a-12B6H2 No 2

TABLE 40 Concentration of humanised bivalent nanobody after heating atincreasing temperatures Nanobody Tags 37° C. 50° C. 60° C. 70° C. 80° C.90° C. 12A2H1-3a-12A2H1 Yes 98 96 93 63 8 9 12A2H1-3a-12A2H1 No 100 99100 77 14 15 12A2H4-3a-12A2H4 Yes 100 99 95 9 6 9 12A2H4-3a-12A2H4 No 9898 98 18 17 26 12B6H2-3a-12B6H2 Yes 100 91 85 7 6 7 12B6H2-3a-12B6H2 No100 99 99 28 13 18

TABLE 41 Platelet adhesion of wild type and humanised bivalentnanobodies % Platelet adhesion Nanobody 0 μg/ml 0.15 μg/ml 0.3 μg/ml 0.6μg/ml Control 65 ± 8 — — — 12B6-3a-12B6 — 50 ± 5 15 ± 6 8 ± 612B6H2-3a-12B6H2 — 53 ± 4  30 ± 16 17 ± 6  12A2-3a-12A2 — 36 ± 8 10 ± 84 ± 3 12A2H1-3a-12A2H1 — 54 ± 4  10 ± 11 12 ± 7  12A2H4-3a-12A2H4 — 38 ±2 10 ± 6 8 ± 4

TABLE 42 baboons used with the different test compounds in the Foltsstudy Baboon Weight ID Sex [kg] Left leg Right leg  1 male 9.8 Control 2 male 10.0 Control 13 male 12.4 Reopro 14 male 9.5 Reopro 15 male 10.8Reopro 16 male ALX-0081 17 male 15.6 ALX-0081 18 male 17.2 ALX-008120/22  male 12.7 ALX-0081 ALX-0081 21 female 8.0 ALX-0081 23 maleALX-0081 24 male 9.4 ALX-0081 3/19 male 15.2 Aspegic ALX-0081  4 female13.6 Aspegic  5 male 17.4 Aspegic  9 male 13.2 Plavix 6/10 male 10.2Heparin Plavix 7/11 male 9.4 Heparin Plavix 8/12 male 10.5 HeparinPlavix

TABLE 43 Length of CFRs (s) for control animals (ND = not done) ControlBaboon ID Final dose Dose 1 2 0 control 291 249 0 saline 294 278 0saline 427 185 0 saline 285 203 0 saline 438 175

TABLE 44 Length of CFRs (s) for animals treated with Aspegic ™ (ND = notdone) Aspegic Baboon ID Final dose Dose 3 4 5 0 control 88 no CFRs 148 0saline 147 204 184 1 mg/kg  1 mg/kg 149 164 135 2.5 mg/kg  1.5 mg/kg 102 325 115 5 mg/kg 2.5 mg/kg  102 1800 245 10 mg/kg  5 mg/kg 113 905156 20 mg/kg 10 mg/kg 125 657 169 40 mg/kg 20 mg/kg 110 ND 145 80 mg/kg40 mg/kg 129 ND ND Epinephrin ND 161 ND

TABLE 45 Length of CFRs (s) for animals treated with Heparin ™ (ND = notdone) Heparin Baboon ID Final dose Dose 6 7 8 0 control 232 113 166 0saline 298 131 246 15 IU/kg 15 IU/kg 630 208 255 30 IU/kg 30 IU/kg 355241 320 60 IU/kg 60 IU/kg 432 246 332 120 IU/kg 120 IU/kg  610 160 206240 IU/kg 240 IU/kg  >1800 221 169 Epinephrin 109 65 ND

TABLE 46 Length of CFRs (s) for animals treated with Plavix ™ (ND = notdone) Plavix Baboon ID Final dose Dose 9 10 11 12   0 control 215 178 84144   0 saline 168 160 88 189   1 mg/kg   1 mg/kg 189 ND 132 179 2.5mg/kg 2.5 mg/kg 883 400 258 >1800   5 mg/kg 2.5mg/kg >1800 >1800 >1800 >1800  10 mg/kg   5mg/kg >1800 >1800 >1800 >1800  20 mg/kg  10mg/kg >1800 >1800 >1800 >1800 Epinephrin 241 91 83 66

TABLE 47 Length of CFRs (s) for animals treated with Reopro ™ (ND = notdone) Reopro Baboon ID Final dose Dose 13 14 15 0 control 144 90 308 0saline 141 103 268 20 μg/kg  20 μg/kg 98 82 254 70 μg/kg  50 μg/kg 90 90248 170 μg/kg 100 μg/kg 90 >1800 >1800 420 μg/kg 250μg/kg >1800 >1800 >1800 920 μg/kg 500 g/kg  >1800 >1800 >1800Epinephrin >1200 >1200 >1200

TABLE 48 Length of CFRs (s) for animals treated with ALX-0081 (ND = notdone) ALX-0081 Baboon ID Final dose Dose 16 17 18 19 20 21 22 23 24  0control 168 293 185 90 94 117 188 164 161  0 saline 151 236 242 117 98107 233 105 178  3 μg/kg  3 micro- 193 ND ND 144 133 183 312 112 295gram/kg  13 μg/kg  10 micro- 913 >1800 298 237 620 525 >1800 213 380gram/kg  43 μg/kg  30micro- >1800 >1800 >1800 >1800 >1800 >1800 >1800 >1800 >1800 gram/kg 133μg/kg  90 micro- >1800 >1800 >1800 >1800 >1800 >1800 >1800 >1800 >1800gram/kg 403 μg/kg 270micro- >1800 >1800 >1800 >1800 >1800 >1800 >1800 >1800 >1800 gram/kgEpinephrin >1200 >1200 >900 >900 >900 >900 >900 >900 >900

TABLE 49 baboons used with the different test compounds in the Foltsstudy Baboon ID Sex Weight [kg] Mix 1 Male 9.8 Asp/Hep/Plav/ALX 2 Female13.6 Asp/Hep/Plav/ALX 3 Female 7.8 Asp/Hep/Plav/ALX 4 Male 12.1Asp/Hep/Plav/ALX 5 Male 11.4 Asp/Hep/Plav/ALX 6 Male 11.4Asp/Hep/Plav/ALX 7 Male 14.0 Asp/Hep/Plav/ALX

TABLE 50 Inhibition of CFRs in the Folts model for the different drugstested. The number of experiments in which an inhibition of CFRs wasobserved in the mentioned different conditions is shown as a function ofthe total number of independent repeats of that condition. Inhibition ofInhibition of CFRs after Test Inhibition CFRs after new administrationEffective compound of CFRs injury of Epinephrin dose Control 0/2 ND ND —Aspegic 0/3 0/3 0/1 — Heparin 1/3 1/3 0/2 — Plavix 4/4 4/4 0/4 5 mg/kgReopro 3/3 3/3 3/3 170-420 μg/kg ALX-0081 9/9 9/9 9/9 13-43 μg/kg + 1.5× dose/kg/hour

TABLE 51 Length of CFRs (seconds) for each baboon and each dose ofAspegic, Heparin, Plavix and ALX-0081. The effective dose is indicatedin yellow 4 5 6 7 8 9 10 control 82 99 109 95 113 142 113 saline 113 119108 114 131 168 92 5 mg/kg Aspegic + 60 IU/kg Heparin + 153 135 272 742146 219 223 1 mg/kg Plavix +1 mg/kgPlavix >1800 >1800 >1800 >1800 >1800 >1800 >1800 Epinephrin + noneffective doses 250 72 87 95 106 105 105 ALX-0081 + Heparin Epinephrin +effective dose ALX-0081 >1800 >1800 >1800 >1800 >1800 >1800 >1800

TABLE 52 Blood loss relative to the second control gauze for animalstreated with Plavix ™ in function of final dose (STD = standarddeviation) Plavix Baboon ID Final dose Dose 9 10 11 12 Average STD   1mg/kg   1 mg/kg 0.6 1.3 1.6 1.2 1.4 0.2 2.5 mg/kg 1.5 mg/kg 1.5 1.4 1.11.0 1.2 0.2   5 mg/kg 2.5 mg/kg 5.1 4.7 1.4 6.6 4.5 2.2  10 mg/kg   5mg/kg 6.5 4.5 5.8 13.6 7.6 4.1  20 mg/kg  10 mg/kg 3.7 4.1 9.1 2.6 4.92.9

TABLE 53 Blood loss relative to the second control gauze for animalstreated with Reopro ™ in function of final dose (STD = standarddeviation) Reopro Baboon ID Final dose Dose 13 14 15 Average STD  20μg/kg  20 μg/kg 2.7 0.6 2.1 1.8 1  70 μg/kg  50 μg/kg 1.2 2.1 0.4 1.2 1170 μg/kg 100 μg/kg 1.2 6.0 4.7 4.0 2 420 μg/kg 250 μg/kg 28.3 7.1 13.916.4 11 920 μg/kg 500 μg/kg 39.8 14.5 6.3 20.2 17

TABLE 54 Blood loss relative to the second control gauze for animalstreated with ALX- 0081 in function of final dose (STD = standarddeviation) ALX-0081 Baboon ID Final dose Dose 17 18 19 20 21 22 23 24Average STD  3 μg/kg  3 μg/kg ND ND 0.4 1.2 0.5 1.1 1.8 0.8 1.0 0.5  13μg/kg  10 μg/kg 1.2 0.8 0.1 1.2 1.1 1.3 1.1 1.1 1.0 0.4  43 μg/kg  30μg/kg 2.8 2.0 4.1 3.9 2.1 3.8 3.0 1.6 2.9 1.0 133 μg/kg  90 μg/kg 2.53.1 2.0 0.4 4.0 5.3 5.6 1.4 3.0 1.8 403 μg/kg 270 μg/kg 2.6 2.3 2.3 0.40.8 5.6 10.7 0.9 3.2 3.4

TABLE 55 The average of the total amount of blood loss (= sum of bloodloss from the first five doses of test compound) as relative to thesecond control gauze Average of Standard Total dose as multiple of totalblood deviation of Test compound effective dose loss blood loss Plavix 419.4 4.0 Reopro 3.5 44 26 ALX-0081 13 10.8 5.7

TABLE 56 Blood loss in gauzes relative to the second control gauze foreach baboon treated with Aspegic, Heparin, Plavix and ALX-0081 infunction of drug dose. The effective drug dose in which a completeinhibition of CFRs was observed, is indicated in yellow median ± 4 5 6 78 9 10 STD 5 mg/kg Aspegic + 60 IU/kg Heparin + 2.6 1 4.6 12.1 1.3 1.41.8 1.8 ± 3.5  1 mg/kg Plavix +1 mg/kg Plavix 3.7 13.9 4.8 13.1 1.4 4.35.6 4.8 ± 6.7  Epinephrin + non effective doses ALX- 9.4 2.7 2.7 67 0.34.8 9.2 4.8 ± 13.7 0081 + Heparin Epinephrin + effective dose ALX-008123.4 2.0 1 39.4 1.3 4.5 0.8 2.0 ± 10.3

TABLE 57 % ristocetin-induced platelet aggregation for each baboontreated with Aspegic, Heparin, Plavix and ALX-0081 in function of drugdose 4 5 6 7 8 9 10 control 75 78 79 70 46 70 46 saline 48 78 74 24 4565 47 5 mg/kg Aspegic + 60 IU/kg 62 65 89 64 66 68 55 Heparin + 1 mg/kgPlavix +1 mg/kg Plavix 42 63 66 83 59 76 60 Epinephrin + effective doseALX-0081 0 24 24 6 17 7 8

TABLE 58 concentration of ALX-0081 [μg/ml] in blood samples obtained at10 minutes after administration ALX-0081 Baboon ID Final dose Dose 16 1718 19 20 21 22 23 24  3 μg/kg  3 μg/kg 0.10 0.03 0.05 0.08 0.04 0.140.08  13 μg/kg  10 μg/kg 1.23 0.34 0.29 0.26 0.50 0.39 0.41 0.18 0.42 43 μg/kg  30 μg/kg 1.00 0.51 0.72 1.14 1.01 0.61 1.01 0.87 1.21 133μg/kg  90 μg/kg 1.87 1.38 1.77 1.61 2.64 1.60 6.77 2.75 5.01 403 μg/kg270 μg/kg 6.77 4.03 6.73 35.14 7.66 6.01 9.24 18.56 16.62

TABLE 59 Length of CFRs [seconds] for baboons treated with ALX-0081 andwith vWF Baboon ID Dose 1 2 3 Control 119 140 111 Saline ND 158 158  30μg/kg + 45 μg/kg/hour ALX-0081 >1800 >1800 >1800 250 IUvWF >420 >1800 >1800 250 IU vWF 246 256 230

TABLE 60 Volumes [μl] to prepare the different mixtures for study ofcleavage of A1A2A3 by ADAMTS13 NPP + NPP NPP + EDTA ALX-0081 PBS Tris(100 mM) 5 5 5 5 BaCl₂ (10 mM) 5 5 5 5 Pefablock (100 mM) 1.3 1.3 1.31.3 Plasma 3.3 3.3 3.3 3.3 ALX 0081 (2.48 mg/ml) — — 4 — PBS — — — 4EDTA (0.35M) pH = 8.3 — 2.6 — — H₂O 81.9 79.3 77.9 77.9 A1A2A3 (460g/ml) 3.5 3.5 3.5 3.5

1. Protein or polypeptide comprising SEQ ID NO:98.
 2. A pharmaceuticalcomposition, comprising a protein or polypeptide according to claim 1,and optionally at least one pharmaceutically acceptable carrier.
 3. Anucleotide sequence or nucleic acid, encoding a protein or polypeptideaccording to claim
 1. 4. A host cell, comprising a nucleotide sequenceor nucleic acid according to claim
 3. 5. A host cell which expresses oris capable of expressing a protein, or polypeptide according to claim 1.6. A method for preparing a protein or polypeptide according to claim 1,which comprises cultivating or maintaining a host cell under conditionssuch that said host cell produces or expresses a protein, or polypeptideaccording to claim 1; and which optionally further comprises isolatingthe protein, or polypeptide according to claim 1.